Microorganisms for the production of adipic acid and other compounds

ABSTRACT

The invention provides a non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid or caprolactam pathway. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in the respective adipate, 6-aminocaproic acid or caprolactam pathway. The invention additionally provides a method for producing adipate, 6-aminocaproic acid or caprolactam. The method can include culturing an adipate, 6-aminocaproic acid or caprolactam producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an adipate, 6-aminocaproic acid or caprolactam pathway enzyme in a sufficient amount to produce the respective product, under conditions and for a sufficient period of time to produce adipate, 6-aminocaproic acid or caprolactam.

This application is a continuation of U.S. application Ser. No.15/184,940, filed Jun. 16, 2016, which is a continuation of U.S.application Ser. No. 14/450,150, filed Aug. 1, 2014, now U.S. Pat. No.9,382,556, issued Jul. 5, 2016, which is a continuation of U.S.application Ser. No. 13/452,642, filed Apr. 20, 2012, which is acontinuation of U.S. application Ser. No. 13/288,699, filed Nov. 3,2011, now U.S. Pat. No. 8,216,814, issued Jul. 10, 2012, which is acontinuation of U.S. application Ser. No. 13/088,256, filed Apr. 15,2011, now U.S. Pat. No. 8,062,871, issued Nov. 22, 2011, which is acontinuation of U.S. application Ser. No. 12/875,084, filed Sep. 2,2010, now U.S. Pat. No. 8,088,607, issued Jan. 3, 2012, which is acontinuation of U.S. application Ser. No. 12/413,355, filed Mar. 27,2009, now U.S. Pat. No. 7,799,545, issued Sep. 21, 2010, which claimsthe benefit of priority of U.S. Provisional Ser. No. 61/040,059, filedMar. 27, 2008, each of which the entire contents are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biosynthetic processes, andmore specifically to organisms having adipic acid, 6-aminocaproic acidand caprolactam biosynthetic capability.

Adipic acid, a dicarboxylic acid, with molecular weight of 146.14, is acompound of commercial significance. Its major use is to produce nylon6,6, a linear polyamide made by condensing adipic acid withhexamethylene diamine that is primarily employed for manufacturingdifferent kinds of fibers. Other uses of adipic acid include its use inplasticizers, unsaturated polyesters, and polyester polyols. Additionaluses include for production of polyurethane, lubricant components, andas a food ingredient as a flavorant and gelling aid.

Historically, adipic acid was prepared from various fats usingoxidation. The current commercial processes for adipic acid synthesisrely on the oxidation of KA oil, a mixture of cyclohexanone, the ketoneor K component, and cyclohexanol, the alcohol or A component, or of purecyclohexanol using an excess of strong nitric acid. There are severalvariations of this theme which differ in the routes for production of KAor cyclohexanol. For example, phenol is an alternative raw material inKA oil production, and the process for the synthesis of adipic acid fromphenol has been described. The other versions of this process tend touse oxidizing agents other than nitric acid, such as hydrogen peroxide,air or oxygen.

Caprolactam is an organic compound which is a lactam of 6-aminohexanoicacid (ε-aminohexanoic acid, aminocaproic acid). It can alternatively beconsidered cyclic amide of caproic acid. The primary industrial use ofcaprolactam is as a monomer in the production of nylon-6. Most of thecaprolactam is synthesised from cyclohexanone via an oximation processusing hydroxylammonium sulfate followed by catalytic rearrangement usingthe Beckmann rearrangement process step.

Thus, there exists a need for alternative methods for effectivelyproducing commercial quantities of compounds such as adipic acid andcarpolactam. The present invention satisfies this need and providesrelated advantages as well.

SUMMARY OF INVENTION

The invention provides a non-naturally occurring microbial organismhaving an adipate, 6-aminocaproic acid or caprolactam pathway. Themicrobial organism contains at least one exogenous nucleic acid encodingan enzyme in the respective adipate, 6-aminocaproic acid or caprolactampathway. The invention additionally provides a method for producingadipate, 6-aminocaproic acid or caprolactam. The method can includeculturing an adipate, 6-aminocaproic acid or caprolactam producingmicrobial organism, where the microbial organism expresses at least oneexogenous nucleic acid encoding an adipate, 6-aminocaproic acid orcaprolactam pathway enzyme in a sufficient amount to produce therespective product, under conditions and for a sufficient period of timeto produce adipate, 6-aminocaproic acid or caprolactam.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary pathway for adipate degradation in theperoxisome of Penicillium chrysogenum.

FIG. 2 shows an exemplary pathway for adipate formation via a reversedegradation pathway. Several options are provided for the finalconversion of adipyl-CoA to adipate.

FIG. 3 shows an exemplary pathway for adipate formation via the3-oxoadipate pathway.

FIG. 4 show the similar enzyme chemistries of the last three steps ofthe 3-oxoadipate pathway for adipate synthesis and the reductive TCAcycle.

FIG. 5 shows an exemplary pathway for synthesis of adipic acid fromglucose via cis,cis-muconic acid. Biosynthetic intermediates(abbreviations): D-erythrose 4-phosphate (E4P), phosphoenolpyruvic acid(PEP), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP),3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), protocatechuicacid (PCA). Enzymes (encoding genes) or reaction conditions: (a) DAHPsynthase (aroFFBR), (b) 3-dehydroquinate synthase (aroB), (c)3-dehydroquinate dehydratase (aroD), (d) DHS dehydratase (aroZ), (e)protocatechuate decarboxylase (aroY), (f) catechol 1,2-dioxygenase(catA), (g) 10% Pt/C, H₂, 3400 kPa, 25° C. Figure taken from Niu et al.,Biotechnol. Prog. 18:201-211 (2002)).

FIG. 6 shows an exemplary pathway for adipate synthesis viaalpha-ketoadipate using alpha-ketoglutarate as a starting point.

FIG. 7 shows an exemplary pathway for synthesis of adipate using lysineas a starting point.

FIG. 8 shows an exemplary caprolactam synthesis pathway using adipyl-CoAas a starting point.

FIG. 9 shows exemplary adipate synthesis pathways usingalpha-ketoadipate as a starting point.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the design and production of cellsand organisms having biosynthetic production capabilities for adipate,6-aminocaproic acid or caprolactam. The results described hereinindicate that metabolic pathways can be designed and recombinantlyengineered to achieve the biosynthesis of adipate, 6-aminocaproic acidor caprolactam in Escherichia coli and other cells or organisms.Biosynthetic production of adipate, 6-aminocaproic acid and caprolactamcan be confirmed by construction of strains having the designedmetabolic genotype. These metabolically engineered cells or organismsalso can be subjected to adaptive evolution to further augment adipate,6-aminocaproic acid or caprolactam biosynthesis, including underconditions approaching theoretical maximum growth.

As disclosed herein, a number of metabolic pathways for the productionof adipate, 6-aminocaproate, and caprolactam are described. Two routes,the reverse adipate degradation pathway and the 3-oxoadipate pathway,were found to be beneficial with respect to (i) the adipate yields (92%molar yield on glucose), (ii) the lack of oxygen requirement for adipatesynthesis, (iii) the associated energetics, and (iv) the theoreticalcapability to produce adipate as the sole fermentation product.Metabolic pathways for adipate production that pass throughα-ketoadipate or lysine are also described but are lower yielding andrequire aeration for maximum production. A pathway for producing eitheror both of 6-aminocaproate and caprolactam from adipyl-CoA, a precursorin the reverse degradation pathway, is also disclosed herein.

As disclosed herein, a number of exemplary pathways for biosynthesis ofadipate are described. One exemplary pathway involves adipate synthesisvia a route that relies on the reversibility of adipate degradation asdescribed in organisms such as P. chrysogenum (see Examples I and II). Asecond exemplary pathway entails the formation of 3-oxoadipate followedby its reduction, dehydration and again reduction to form adipate (seeExamples III and IV). The adipate yield using either of these twopathways is 0.92 moles per mole glucose consumed. The uptake of oxygenis not required for attaining these theoretical maximum yields, and theenergetics under anaerobic conditions are favorable for growth andproduct secretion. A method for producing adipate from glucose-derivedcis,cis-muconic acid was described previously (Frost et al., U.S. Pat.No. 5,487,987, issued Jan. 30, 1996) (see Example V). Advantages of theembodiments disclosed herein over this previously described method arediscussed. Metabolic pathways for adipate production that pass throughα-ketoadipate (Example VI) or lysine (Example VII) precursors are loweryielding and require aeration for maximum production. A pathway forproducing either or both of 6-aminocaproate and caprolactam fromadipyl-CoA, a precursor in the reverse degradation pathway, is described(see Example VIII and IX). Additional pathways for producing adipate aredescribed in Examples X and XI. Exemplary genes and enzymes required forconstructing microbes with these capabilities are described as well asmethods for cloning and transformation, monitoring product formation,and using the engineered microorganisms for production.

As disclosed herein, six different pathways for adipic acid synthesisusing glucose/sucrose as a carbon substrate are described. For allmaximum yield calculations, the missing reactions in a given pathwaywere added to the E. coli stoichiometric network in SimPheny that issimilar to the one described previously (Reed et al., Genome Biol. 4:R54(2003)). Adipate is a charged molecule under physiological conditionsand was assumed to require energy in the form of a proton-based symportsystem to be secreted out of the network. Such a transport system isthermodynamically feasible if the fermentations are carried out atneutral or near-neutral pH. Low pH adipic acid formation would requirean ATP-dependant export mechanism, for example, the ABC system asopposed to proton symport. The reactions in the pathways and methods ofimplementation of these pathways are described in Examples I-XI.

As used herein, the term “non-naturally occurring” when used inreference to a microbial organism or microorganism of the invention isintended to mean that the microbial organism has at least one geneticalteration not normally found in a naturally occurring strain of thereferenced species, including wild-type strains of the referencedspecies. Genetic alterations include, for example, modificationsintroducing expressible nucleic acids encoding metabolic polypeptides,other nucleic acid additions, nucleic acid deletions and/or otherfunctional disruption of the microbial genetic material. Suchmodifications include, for example, coding regions and functionalfragments thereof, for heterologous, homologous or both heterologous andhomologous polypeptides for the referenced species. Additionalmodifications include, for example, non-coding regulatory regions inwhich the modifications alter expression of a gene or operon. Exemplarymetabolic polypeptides include enzymes within an adipate, 6-aminocaproicacid or caprolactam biosynthetic pathway.

A metabolic modification refers to a biochemical reaction that isaltered from its naturally occurring state. Therefore, non-naturallyoccurring microorganisms can have genetic modifications to nucleic acidsencoding metabolic polypeptides or, functional fragments thereof.Exemplary metabolic modifications are disclosed herein.

As used herein, the term “isolated” when used in reference to amicrobial organism is intended to mean an organism that is substantiallyfree of at least one component as the referenced microbial organism isfound in nature. The term includes a microbial organism that is removedfrom some or all components as it is found in its natural environment.The term also includes a microbial organism that is removed from some orall components as the microbial organism is found in non-naturallyoccurring environments. Therefore, an isolated microbial organism ispartly or completely separated from other substances as it is found innature or as it is grown, stored or subsisted in non-naturally occurringenvironments. Specific examples of isolated microbial organisms includepartially pure microbes, substantially pure microbes and microbescultured in a medium that is non-naturally occurring.

As used herein, the terms “microbial,” “microbial organism” or“microorganism” is intended to mean any organism that exists as amicroscopic cell that is included within the domains of archaea,bacteria or eukarya. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. The term alsoincludes cell cultures of any species that can be cultured for theproduction of a biochemical.

As used herein, the term “CoA” or “coenzyme A” is intended to mean anorganic cofactor or prosthetic group (nonprotein portion of an enzyme)whose presence is required for the activity of many enzymes (theapoenzyme) to form an active enzyme system. Coenzyme A functions incertain condensing enzymes, acts in acetyl or other acyl group transferand in fatty acid synthesis and oxidation, pyruvate oxidation and inother acetylation.

As used herein, “adipate,” having the chemical formula —OOC—(CH₂)₄—COO—(see FIG. 2) (IUPAC name hexanedioate), is the ionized form of adipicacid (IUPAC name hexanedioic acid), and it is understood that adipateand adipic acid can be used interchangeably throughout to refer to thecompound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled understand that thespecific form will depend on the pH.

As used herein, “6-aminocaproate,” having the chemical formula —OOC—(CH₂)₅—NH₂ (see FIG. 8), is the ionized form of 6-aminocaproic acid(IUPAC name 6-aminohexanoic acid), and it is understood that6-aminocaproate and 6-aminocaproic acid can be used interchangeablythroughout to refer to the compound in any of its neutral or ionizedforms, including any salt forms thereof. It is understood by thoseskilled understand that the specific form will depend on the pH.

As used herein, “caprolactam” (IUPAC name azepan-2-one) is a lactam of6-aminohexanoic acid (see FIG. 8).

As used herein, the term “substantially anaerobic” when used inreference to a culture or growth condition is intended to mean that theamount of oxygen is less than about 10% of saturation for dissolvedoxygen in liquid media. The term also is intended to include sealedchambers of liquid or solid medium maintained with an atmosphere of lessthan about 1% oxygen.

“Exogenous” as it is used herein is intended to mean that the referencedmolecule or the referenced activity is introduced into the hostmicrobial organism. The molecule can be introduced, for example, byintroduction of an encoding nucleic acid into the host genetic materialsuch as by integration into a host chromosome or as non-chromosomalgenetic material such as a plasmid. Therefore, the term as it is used inreference to expression of an encoding nucleic acid refers tointroduction of the encoding nucleic acid in an expressible form intothe microbial organism. When used in reference to a biosyntheticactivity, the term refers to an activity that is introduced into thehost reference organism. The source can be, for example, a homologous orheterologous encoding nucleic acid that expresses the referencedactivity following introduction into the host microbial organism.Therefore, the term “endogenous” refers to a referenced molecule oractivity that is present in the host. Similarly, the term when used inreference to expression of an encoding nucleic acid refers to expressionof an encoding nucleic acid contained within the microbial organism. Theterm “heterologous” refers to a molecule or activity derived from asource other than the referenced species whereas “homologous” refers toa molecule or activity derived from the host microbial organism.Accordingly, exogenous expression of an encoding nucleic acid of theinvention can utilize either or both a heterologous or homologousencoding nucleic acid.

The non-naturally occurring microbal organisms of the invention cancontain stable genetic alterations, which refers to microorganisms thatcan be cultured for greater than five generations without loss of thealteration. Generally, stable genetic alterations include modificationsthat persist greater than 10 generations, particularly stablemodifications will persist more than about 25 generations, and moreparticularly, stable genetic modifications will be greater than 50generations, including indefinitely.

Those skilled in the art will understand that the genetic alterations,including metabolic modifications exemplified herein, are described withreference to a suitable host organism such as E. coli and theircorresponding metabolic reactions or a suitable source organism fordesired genetic material such as genes for a desired metabolic pathway.However, given the complete genome sequencing of a wide variety oforganisms and the high level of skill in the area of genomics, thoseskilled in the art will readily be able to apply the teachings andguidance provided herein to essentially all other organisms. Forexample, the E. coli metabolic alterations exemplified herein canreadily be applied to other species by incorporating the same oranalogous encoding nucleic acid from species other than the referencedspecies. Such genetic alterations include, for example, geneticalterations of species homologs, in general, and in particular,orthologs, paralogs or nonorthologous gene displacements.

An ortholog is a gene or genes that are related by vertical descent andare responsible for substantially the same or identical functions indifferent organisms. For example, mouse epoxide hydrolase and humanepoxide hydrolase can be considered orthologs for the biologicalfunction of hydrolysis of epoxides. Genes are related by verticaldescent when, for example, they share sequence similarity of sufficientamount to indicate they are homologous, or related by evolution from acommon ancestor. Genes can also be considered orthologs if they sharethree-dimensional structure but not necessarily sequence similarity, ofa sufficient amount to indicate that they have evolved from a commonancestor to the extent that the primary sequence similarity is notidentifiable. Genes that are orthologous can encode proteins withsequence similarity of about 25% to 100% amino acid sequence identity.Genes encoding proteins sharing an amino acid similarity less that 25%can also be considered to have arisen by vertical descent if theirthree-dimensional structure also shows similarities. Members of theserine protease family of enzymes, including tissue plasminogenactivator and elastase, are considered to have arisen by verticaldescent from a common ancestor.

Orthologs include genes or their encoded gene products that through, forexample, evolution, have diverged in structure or overall activity. Forexample, where one species encodes a gene product exhibiting twofunctions and where such functions have been separated into distinctgenes in a second species, the three genes and their correspondingproducts are considered to be orthologs. For the production of abiochemical product, those skilled in the art will understand that theorthologous gene harboring the metabolic activity to be introduced ordisrupted is to be chosen for construction of the non-naturallyoccurring microorganism. An example of orthologs exhibiting separableactivities is where distinct activities have been separated intodistinct gene products between two or more species or within a singlespecies. A specific example is the separation of elastase proteolysisand plasminogen proteolysis, two types of serine protease activity, intodistinct molecules as plasminogen activator and elastase. A secondexample is the separation of mycoplasma 5′-3′ exonuclease and DrosophilaDNA polymerase III activity. The DNA polymerase from the first speciescan be considered an ortholog to either or both of the exonuclease orthe polymerase from the second species and vice versa.

In contrast, paralogs are homologs related by, for example, duplicationfollowed by evolutionary divergence and have similar or common, but notidentical functions. Paralogs can originate or derive from, for example,the same species or from a different species. For example, microsomalepoxide hydrolase (epoxide hydrolase I) and soluble epoxide hydrolase(epoxide hydrolase II) can be considered paralogs because they representtwo distinct enzymes, co-evolved from a common ancestor, that catalyzedistinct reactions and have distinct functions in the same species.Paralogs are proteins from the same species with significant sequencesimilarity to each other suggesting that they are homologous, or relatedthrough co-evolution from a common ancestor. Groups of paralogousprotein families include HipA homologs, luciferase genes, peptidases,and others.

A nonorthologous gene displacement is a nonorthologous gene from onespecies that can substitute for a referenced gene function in adifferent species. Substitution includes, for example, being able toperform substantially the same or a similar function in the species oforigin compared to the referenced function in the different species.Although generally, a nonorthologous gene displacement will beidentifiable as structurally related to a known gene encoding thereferenced function, less structurally related but functionally similargenes and their corresponding gene products nevertheless will still fallwithin the meaning of the term as it is used herein. Functionalsimilarity requires, for example, at least some structural similarity inthe active site or binding region of a nonorthologous gene productcompared to a gene encoding the function sought to be substituted.Therefore, a nonorthologous gene includes, for example, a paralog or anunrelated gene.

Therefore, in identifying and constructing the non-naturally occurringmicrobial organisms of the invention having adipate, 6-aminocaproic acidor caprolactam biosynthetic capability, those skilled in the art willunderstand with applying the teaching and guidance provided herein to aparticular species that the identification of metabolic modificationscan include identification and inclusion or inactivation of orthologs.To the extent that paralogs and/or nonorthologous gene displacements arepresent in the referenced microorganism that encode an enzyme catalyzinga similar or substantially similar metabolic reaction, those skilled inthe art also can utilize these evolutionally related genes.

Orthologs, paralogs and nonorthologous gene displacements can bedetermined by methods well known to those skilled in the art. Forexample, inspection of nucleic acid or amino acid sequences for twopolypeptides will reveal sequence identity and similarities between thecompared sequences. Based on such similarities, one skilled in the artcan determine if the similarity is sufficiently high to indicate theproteins are related through evolution from a common ancestor.Algorithms well known to those skilled in the art, such as Align, BLAST,Clustal W and others compare and determine a raw sequence similarity oridentity, and also determine the presence or significance of gaps in thesequence which can be assigned a weight or score. Such algorithms alsoare known in the art and are similarly applicable for determiningnucleotide sequence similarity or identity. Parameters for sufficientsimilarity to determine relatedness are computed based on well knownmethods for calculating statistical similarity, or the chance of findinga similar match in a random polypeptide, and the significance of thematch determined. A computer comparison of two or more sequences can, ifdesired, also be optimized visually by those skilled in the art. Relatedgene products or proteins can be expected to have a high similarity, forexample, 25% to 100% sequence identity. Proteins that are unrelated canhave an identity which is essentially the same as would be expected tooccur by chance, if a database of sufficient size is scanned (about 5%).Sequences between 5% and 24% may or may not represent sufficienthomology to conclude that the compared sequences are related. Additionalstatistical analysis to determine the significance of such matches giventhe size of the data set can be carried out to determine the relevanceof these sequences.

Exemplary parameters for determining relatedness of two or moresequences using the BLAST algorithm, for example, can be as set forthbelow. Briefly, amino acid sequence alignments can be performed usingBLASTP version 2.0.8 (Jan. 5, 1999) and the following parameters:Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50;expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignmentscan be performed using BLASTN version 2.0.6 (Sep. 16, 1998) and thefollowing parameters: Match: 1; mismatch: −2; gap open: 5; gapextension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off.Those skilled in the art will know what modifications can be made to theabove parameters to either increase or decrease the stringency of thecomparison, for example, and determine the relatedness of two or moresequences.

The invention provides non-naturally occurring microbial organismscapable of producing adipate, 6-aminocaproic acid or caprolactam. Forexample, an adipate pathway can be a reverse adipate degradation pathway(see Examples I and II). In one embodiment, the invention provides anon-naturally occurring microbial organism having an adipate pathwaycomprising at least one exogenous nucleic acid encoding an adipatepathway enzyme expressed in a sufficient amount to produce adipate, theadipate pathway comprising succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase orphosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase. In addition, an adipate pathway canbe through a 3-oxoadipate pathway (see Examples III and IV). In anotherembodiment, the invention provides a non-naturally occurring microbialorganism having an adipate pathway comprising at least one exogenousnucleic acid encoding an adipate pathway enzyme expressed in asufficient amount to produce adipate, the adipate pathway comprisingsuccinyl-CoA:acetyl-CoA acyl transferase, 3-oxoadipyl-CoA transferase,3-oxoadipate reductase, 3-hydroxyadipate dehydratase, and 2-enoatereductase.

In still another embodiment, the invention provides a non-naturallyoccurring microbial organism having a 6-aminocaproic acid pathwaycomprising at least one exogenous nucleic acid encoding a 6-aminocaproicacid pathway enzyme expressed in a sufficient amount to produce6-aminocaproic acid, the 6-aminocaproic acid pathway comprisingCoA-dependent aldehyde dehydrogenase and transaminase (see Examples VIIIand IX). Alternatively, 6-aminocaproate dehydrogenase can be used toconvert adipate semialdehyde to form 6-aminocaproate (see FIG. 8). In afurther embodiment, the invention provides a non-naturally occurringmicrobial organism having a caprolactam pathway comprising at least oneexogenous nucleic acid encoding a caprolactam pathway enzyme expressedin a sufficient amount to produce caprolactam, the caprolactam pathwaycomprising CoA-dependent aldehyde dehydrogenase, transaminase or6-aminocaproate dehydrogenase, and amidohydrolase (see Examples VIII andIX).

As disclosed herein, a 6-aminocaproic acid or caprolactam producingmicrobial organism of the invention can produce 6-aminocaproic acidand/or caprolactam from an adipyl-CoA precursor (see FIG. 8 and ExamplesVIII and IX). Therefore, it is understood that a 6-aminocaproic acid orcaprolactam producing microbial organism can further include a pathwayto produce adipyl-CoA. For example an adipyl-CoA pathway can include theenzymes of FIG. 2 that utilize succinyl-CoA and acetyl-CoA as precursorsthrough the production of adipyl-CoA, that is, lacking an enzyme for thefinal step of converting adipyl-CoA to adipate. Thus, one exemplaryadipyl-CoA pathway can include succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase and5-carboxy-2-pentenoyl-CoA reductase.

In addition, as shown in FIG. 1, an adipate degradation pathway includesthe step of converting adipate to adipyl-CoA by an adipate CoA ligase.Therefore, an adipyl-CoA pathway can be an adipate pathway that furtherincludes an enzyme activity that converts adipate to adipyl-CoA,including, for example, adipate-CoA ligase activity as in the first stepof FIG. 1 or any of the enzymes in the final step of FIG. 2 carried outin the reverse direction, for example, any of adipyl-CoA synthetase(also referred to as adipate Co-A ligase), phosphotransadipylase/adipatekinase, adipyl-CoA:acetyl-CoA transferase or adipyl-CoA hydrolase. Anenzyme having adipate to adipyl-CoA activity can be an endogenousactivity or can be provided as an exogenous nucleic acid encoding theenzyme, as disclosed herein. Thus, it is understood that any adipatepathway can be utilized with an adipate to adipyl-CoA enzymatic activityto generate an adipyl-CoA pathway. Such a pathway can be included in a6-aminocaproic acid or caprolactam producing microbial organism toprovide an adipyl-CoA precursor for 6-aminocaproic acid and/orcaprolactam production.

An additional exemplary adipate pathway utilizes alpha-ketoadipate as aprecursor (see FIG. 6 and Example VI). In yet another embodiment, theinvention provides a non-naturally occurring microbial organism havingan adipate pathway comprising at least one exogenous nucleic acidencoding an adipate pathway enzyme expressed in a sufficient amount toproduce adipate, the adipate pathway comprising homocitrate synthase,homoaconitase, homoisocitrate dehydrogenase, 2-ketoadipate reductase,alpha-hydroxyadipate dehydratase and oxidoreductase. A further exemplaryadipate pathway utilizes a lysine dedgradation pathway (see FIG. 7 andExample VII). Another embodiment of the invention provides anon-naturally occurring microbial organism having an adipate pathwaycomprising at least one exogenous nucleic acid encoding an adipatepathway enzyme expressed in a sufficient amount to produce adipate, theadipate pathway comprising carbon nitrogen lyase, oxidoreductase,transaminase and oxidoreductase.

Yet another exemplary adipate pathway utilizes alpha-ketoadipate as aprecursor (see FIG. 9 and Examples X and XI). Thus, the inventionadditionally provides a non-naturally occurring microbial organismhaving an adipate pathway comprising at least one exogenous nucleic acidencoding an adipate pathway enzyme expressed in a sufficient amount toproduce adipate, the adipate pathway comprising alpha-ketoadipyl-CoAsynthetase, phosphotransketoadipylase/alpha-ketoadipate kinase oralpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydrogenase; 2-hydroxyadipyl-CoA dehydratase;5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferaseor adipyl-CoA hydrolase. In still another embodiment, the inventionprovides a non-naturally occurring microbial organism having an adipatepathway comprising at least one exogenous nucleic acid encoding anadipate pathway enzyme expressed in a sufficient amount to produceadipate, the adipate pathway comprising 2-hydroxyadipate dehydrogenase;2-hydroxyadipyl-CoA synthetase,phosphotranshydroxyadipylase/2-hydroxyadipate kinase or2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoAsynthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase.

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having an adipate, 6-aminocaproic acid orcaprolactam pathway, wherein the non-naturally occurring microbialorganism comprises at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product, as disclosed herein.Thus, the invention provides a non-naturally occurring microbialorganism containing at least one exogenous nucleic acid encoding apolypeptide, where the polypeptide is an enzyme or protein that convertsthe substrates and products of an adipate, 6-aminocaproic acid orcaprolactam pathway, such as that shown in FIGS. 2, 3, 8 and 9.

In one embodiment, the invention provides a non-naturally occurringmicrobial organism having an adipate pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromsuccinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to3-hydroxyadipyl-CoA; 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;5-carboxy-2-pentenoyl-CoA to adipyl-CoA; adipyl-CoA to adipate (see FIG.2). In another embodiment, the invention provides a non-naturallyoccurring microbial organism having an adipate pathway, wherein themicrobial organism contains at least one exogenous nucleic acid encodinga polypeptide that converts a substrate to a product selected fromsuccinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA; 3-oxoadipyl-CoA to3-oxoadipate; 3-oxoadipate to 3-hydroxyadipate; 3-hydroxyadipate tohexa-2-enedioate; hexa-2-enedioate to adipate (see FIG. 3).

In an additional embodiment, the invention provides a non-naturallyoccurring microbial organism having a 6-aminocaproic acid pathway,wherein the microbial organism contains at least one exogenous nucleicacid encoding a polypeptide that converts a substrate to a productselected from adipyl-CoA to adipate semialdehyde; and adipatesemialdehyde to 6-aminocaproate (see FIG. 8). In still anotherembodiment, the invention provides a non-naturally occurring microbialorganism having a caprolactam pathway, wherein the microbial organismcontains at least one exogenous nucleic acid encoding a polypeptide thatconverts a substrate to a product selected from adipyl-CoA to adipatesemialdehyde; adipate semialdehyde to 6-aminocaproate; and6-aminocaproate to caprolactam.

In still another embodiment, the invention provides a non-naturallyoccurring microbial organism having an adipate pathway, wherein themicrobial organism contains at least one exogenous nucleic acid encodinga polypeptide that converts a substrate to a product selected fromalpha-ketoadipate to alpha-ketoadipyl-CoA; alpha-ketoadipyl-CoA to2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate (seeFIG. 9). Additionally, the invention provides a non-naturally occurringmicrobial organism having an adipate pathway, wherein the microbialorganism contains at least one exogenous nucleic acid encoding apolypeptide that converts a substrate to a product selected fromalpha-ketoadipate to 2-hydroxyadipate; 2-hydroxyadipate to2-hydroxyadipyl-CoA; 2-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA;5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and adipyl-CoA to adipate (FIG.9).

The invention is described herein with general reference to themetabolic reaction, reactant or product thereof, or with specificreference to one or more nucleic acids or genes encoding an enzymeassociated with or catalyzing the referenced metabolic reaction,reactant or product. Unless otherwise expressly stated herein, thoseskilled in the art will understand that reference to a reaction alsoconstitutes reference to the reactants and products of the reaction.Similarly, unless otherwise expressly stated herein, reference to areactant or product also references the reaction, and reference to anyof these metabolic constituents also references the gene or genesencoding the enzymes that catalyze the referenced reaction, reactant orproduct. Likewise, given the well known fields of metabolicbiochemistry, enzymology and genomics, reference herein to a gene orencoding nucleic acid also constitutes a reference to the correspondingencoded enzyme and the reaction it catalyzes as well as the reactantsand products of the reaction.

The non-naturally occurring microbial organisms of the invention can beproduced by introducing expressible nucleic acids encoding one or moreof the enzymes participating in one or more adipate, 6-aminocaproic acidor caprolactam biosynthetic pathways. Depending on the host microbialorganism chosen for biosynthesis, nucleic acids for some or all of aparticular adipate, 6-aminocaproic acid or caprolactam biosyntheticpathway can be expressed. For example, if a chosen host is deficient inone or more enzymes for a desired biosynthetic pathway, then expressiblenucleic acids for the deficient enzyme(s) are introduced into the hostfor subsequent exogenous expression. Alternatively, if the chosen hostexhibits endogenous expression of some pathway genes, but is deficientin others, then an encoding nucleic acid is needed for the deficientenzyme(s) to achieve adipate, 6-aminocaproic acid or caprolactambiosynthesis. Thus, a non-naturally occurring microbial organism of theinvention can be produced by introducing exogenous enzyme activities toobtain a desired biosynthetic pathway or a desired biosynthetic pathwaycan be obtained by introducing one or more exogenous enzyme activitiesthat, together with one or more endogenous enzymes, produces a desiredproduct such as adipate, 6-aminocaproic acid or caprolactam.

Depending on the adipate, 6-aminocaproic acid or caprolactambiosynthetic pathway constituents of a selected host microbial organism,the non-naturally occurring microbial organisms of the invention willinclude at least one exogenously expressed adipate, 6-aminocaproic acidor caprolactam pathway-encoding nucleic acid and up to all encodingnucleic acids for one or more adipate, 6-aminocaproic acid orcaprolactam biosynthetic pathways. For example, adipate, 6-aminocaproicacid or caprolactam biosynthesis can be established in a host deficientin a pathway enzyme through exogenous expression of the correspondingencoding nucleic acid. In a host deficient in all enzymes of a adipate,6-aminocaproic acid or caprolactam pathway, exogenous expression of allenzyme in the pathway can be included, although it is understood thatall enzymes of a pathway can be expressed even if the host contains atleast one of the pathway enzymes.

For example, exogenous expression of all enzymes in a pathway forproduction of adipate can be included in a host organism, such assuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase orphosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase. In particular, a host organism cancontain the adipate pathway enzymes succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoAdehydratase, 5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoAsynthetase. Alternatively, a host organism can contain the adipatepathway enzymes succinyl-CoA:acetyl-CoA acyl transferase,3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and phosphotransadipylase/adipatekinase. In addition, a host organism can contain the adipate pathwayenzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA:acetyl-CoAtransferase. Further, a host organism can contain the adipate pathwayenzymes succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA hydrolase.

In the case of a 6-aminocaproic acid producing microbial organism,exogenous expression of all enzymes in a pathway for production of6-aminocaproic acid can be included in a host organism, such asCoA-dependent aldehyde dehydrogenase and transaminase or CoA-dependentaldehyde dehdrogenase and 6-aminocaproate dehydrogenase. For acaprolactam producing microbial organism, exogenous expression of allenzymes in a pathway for production of caprolactam can be included in ahost organism, such as CoA-dependent aldehyde dehydrogenase,transaminase or 6-aminocaproate dehydrogenase, and amidohydrolase.

Given the teachings and guidance provided herein, those skilled in theart will understand that the number of encoding nucleic acids tointroduce in an expressible form will, at least, parallel the adipate,6-aminocaproic acid or caprolactam pathway deficiencies of the selectedhost microbial organism. Therefore, a non-naturally occurring microbialorganism of the invention can have one, two, three, four, or five, up toall nucleic acids encoding the above enzymes constituting a adipate,6-aminocaproic acid or caprolactam biosynthetic pathway. In someembodiments, the non-naturally occurring microbial organisms also caninclude other genetic modifications that facilitate or optimize adipate,6-aminocaproic acid or caprolactam biosynthesis or that confer otheruseful functions onto the host microbial organism. One such otherfunctionality can include, for example, augmentation of the synthesis ofone or more of the adipate, 6-aminocaproic acid or caprolactam pathwayprecursors such as succinyl-CoA and/or acetyl-CoA in the case of adipatesynthesis, or adipyl-CoA in the case of 6-aminocaproic acid orcaprolactam synthesis, including the adipate pathway enzymes disclosedherein.

In some embodiments, a non-naturally occurring microbial organism of theinvention is generated from a host that contains the enzymaticcapability to synthesize adipate, 6-aminocaproic acid or caprolactam. Inthis specific embodiment it can be useful to increase the synthesis oraccumulation of an adipate, 6-aminocaproic acid or caprolactam pathwayproduct to, for example, drive adipate, 6-aminocaproic acid orcaprolactam pathway reactions toward adipate, 6-aminocaproic acid orcaprolactam production. Increased synthesis or accumulation can beaccomplished by, for example, overexpression of nucleic acids encodingone or more of the above-described adipate, 6-aminocaproic acid orcaprolactam pathway enzymes. Over expression of the adipate,6-aminocaproic acid or caprolactam pathway enzyme or enzymes can occur,for example, through exogenous expression of the endogenous gene orgenes, or through exogenous expression of the heterologous gene orgenes. Therefore, naturally occurring organisms can be readily generatedto be non-naturally occurring microbial organisms of the invention, forexample, producing adipate, 6-aminocaproic acid or caprolactam, throughoverexpression of one, two, three, four, five, that is, up to allnucleic acids encoding adipate, 6-aminocaproic acid or caprolactambiosynthetic pathway enzymes. In addition, a non-naturally occurringorganism can be generated by mutagenesis of an endogenous gene thatresults in an increase in activity of an enzyme in the adipate,6-aminocaproic acid or caprolactam biosynthetic pathway.

In particularly useful embodiments, exogenous expression of the encodingnucleic acids is employed. Exogenous expression confers the ability tocustom tailor the expression and/or regulatory elements to the host andapplication to achieve a desired expression level that is controlled bythe user. However, endogenous expression also can be utilized in otherembodiments such as by removing a negative regulatory effector orinduction of the gene's promoter when linked to an inducible promoter orother regulatory element. Thus, an endogenous gene having a naturallyoccurring inducible promoter can be up-regulated by providing theappropriate inducing agent, or the regulatory region of an endogenousgene can be engineered to incorporate an inducible regulatory element,thereby allowing the regulation of increased expression of an endogenousgene at a desired time. Similarly, an inducible promoter can be includedas a regulatory element for an exogenous gene introduced into anon-naturally occurring microbial organism.

It is understood that, in methods of the invention, any of the one ormore exogenous nucleic acids can be introduced into a microbial organismto produce a non-naturally occurring microbial organism of theinvention. The nucleic acids can be introduced so as to confer, forexample, an adipate, 6-aminocaproic acid or caprolactam biosyntheticpathway onto the microbial organism. Alternatively, encoding nucleicacids can be introduced to produce an intermediate microbial organismhaving the biosynthetic capability to catalyze some of the requiredreactions to confer adipate, 6-aminocaproic acid or caprolactambiosynthetic capability. For example, a non-naturally occurringmicrobial organism having an adipate, 6-aminocaproic acid or caprolactambiosynthetic pathway can comprise at least two exogenous nucleic acidsencoding desired enzymes. In the case of adipate production, the atleast two exogenous nucleic acids can encode the enzymes such as thecombination of succinyl-CoA:acetyl-CoA acyl transferase and3-hydroxyacyl-CoA dehydrogenase, or succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyadipyl-CoA dehydratase, or 3-hydroxyadipyl-CoAand 5-carboxy-2-pentenoyl-CoA reductase, or 3-hydroxyacyl-CoA andadipyl-CoA synthetase, and the like. In the case of caprolactamproduction, the at least two exogenous nucleic acids can encode theenzymes such as the combination of CoA-dependent aldehyde dehydrogenaseand transaminase, or CoA-dependent aldehyde dehydrogenase andamidohydrolase, or transaminase and amidohydrolase. Thus, it isunderstood that any combination of two or more enzymes of a biosyntheticpathway can be included in a non-naturally occurring microbial organismof the invention.

Similarly, it is understood that any combination of three or moreenzymes of a biosynthetic pathway can be included in a non-naturallyoccurring microbial organism of the invention, for example, in the caseof adipate production, the combination of enzymessuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, and 3-hydroxyadipyl-CoA dehydratase; orsuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase and 5-carboxy-2-pentenoyl-CoA reductase; orsuccinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase and adipyl-CoA synthetase; or 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase and adipyl-CoA:acetyl-CoAtransferase, and so forth, as desired, so long as the combination ofenzymes of the desired biosynthetic pathway results in production of thecorresponding desired product. Similarly, any combination of four ormore enzymes of a biosynthetic pathway as disclosed herein can beincluded in a non-naturally occurring microbial organism of theinvention, as desired, so long as the combination of enzymes of thedesired biosynthetic pathway results in production of the correspondingdesired product.

In addition to the biosynthesis of adipate, 6-aminocaproic acid orcaprolactam as described herein, the non-naturally occurring microbialorganisms and methods of the invention also can be utilized in variouscombinations with each other and with other microbial organisms andmethods well known in the art to achieve product biosynthesis by otherroutes. For example, one alternative to produce adipate, 6-aminocaproicacid or caprolactam other than use of the adipate, 6-aminocaproic acidor caprolactam producers is through addition of another microbialorganism capable of converting an adipate, 6-aminocaproic acid orcaprolactam pathway intermediate to adipate, 6-aminocaproic acid orcaprolactam. One such procedure includes, for example, the fermentationof a microbial organism that produces an adipate, 6-aminocaproic acid orcaprolactam pathway intermediate. The adipate, 6-aminocaproic acid orcaprolactam pathway intermediate can then be used as a substrate for asecond microbial organism that converts the adipate, 6-aminocaproic acidor caprolactam pathway intermediate to adipate, 6-aminocaproic acid orcaprolactam. The adipate, 6-aminocaproic acid or caprolactam pathwayintermediate can be added directly to another culture of the secondorganism or the original culture of the adipate, 6-aminocaproic acid orcaprolactam pathway intermediate producers can be depleted of thesemicrobial organisms by, for example, cell separation, and thensubsequent addition of the second organism to the fermentation broth canbe utilized to produce the final product without intermediatepurification steps.

In other embodiments, the non-naturally occurring microbial organismsand methods of the invention can be assembled in a wide variety ofsubpathways to achieve biosynthesis of, for example, adipate,6-aminocaproic acid or caprolactam. In these embodiments, biosyntheticpathways for a desired product of the invention can be segregated intodifferent microbial organisms, and the different microbial organisms canbe co-cultured to produce the final product. In such a biosyntheticscheme, the product of one microbial organism is the substrate for asecond microbial organism until the final product is synthesized. Forexample, the biosynthesis of adipate, 6-aminocaproic acid or caprolactamcan be accomplished by constructing a microbial organism that containsbiosynthetic pathways for conversion of one pathway intermediate toanother pathway intermediate or the product. Alternatively, adipate,6-aminocaproic acid or caprolactam also can be biosynthetically producedfrom microbial organisms through co-culture or co-fermentation using twoorganisms in the same vessel, where the first microbial organismproduces a adipate, 6-aminocaproic acid or caprolactam intermediate andthe second microbial organism converts the intermediate to adipate,6-aminocaproic acid or caprolactam.

Given the teachings and guidance provided herein, those skilled in theart will understand that a wide variety of combinations and permutationsexist for the non-naturally occurring microbial organisms and methods ofthe invention together with other microbial organisms, with theco-culture of other non-naturally occurring microbial organisms havingsubpathways and with combinations of other chemical and/or biochemicalprocedures well known in the art to produce adipate, 6-aminocaproic acidor caprolactam.

Sources of encoding nucleic acids for an adipate, 6-aminocaproic acid orcaprolactam pathway enzyme can include, for example, any species wherethe encoded gene product is capable of catalyzing the referencedreaction. Such species include both prokaryotic and eukaryotic organismsincluding, but not limited to, bacteria, including archaea andeubacteria, and eukaryotes, including yeast, plant, insect, animal, andmammal, including human. Exemplary species for such sources include, forexample, Escherichia coli, Pseudomonas knackmussii, Pseudomonas putida,Pseudomonas fluorescens, Klebsiella pneumoniae, Serratia proteamaculans,Streptomyces sp. 2065, Pseudomonas aeruginosa, Ralstonia eutropha,Clostridium acetobutylicum, Euglena gracilis, Treponema denticola,Clostridium kluyveri, Homo sapiens, Rattus norvegicus, Acinetobacter sp.ADP 1, Streptomyces coelicolor, Eubacterium barkeri, Peptostreptococcusasaccharolyticus, Clostridium botulinum, Clostridium tyrobutyricum,Clostridium thermoaceticum (Moorella thermoaceticum), Acinetobactercalcoaceticus, Mus musculus, Sus scrofa, Flavobacterium sp, Arthrobacteraurescens, Penicillium chrysogenum, Aspergillus niger, Aspergillusnidulans, Bacillus subtilis, Saccharomyces cerevisiae, Zymomonasmobilis, Mannheimia succiniciproducens, Clostridium ljungdahlii,Clostridium carboxydivorans, Geobacillus stearothermophilus,Agrobacterium tumefaciens, Achromobacter denitrificans, Arabidopsisthaliana, Haemophilus influenzae, Acidaminococcus fermentans,Clostridium sp. M62/1, Fusobacterium nucleatum, as well as otherexemplary species disclosed herein or available as source organisms forcorresponding genes (see Examples). However, with the complete genomesequence available for now more than 550 species (with more than half ofthese available on public databases such as the NCBI), including 395microorganism genomes and a variety of yeast, fungi, plant, andmammalian genomes, the identification of genes encoding the requisiteadipate, 6-aminocaproic acid or caprolactam biosynthetic activity forone or more genes in related or distant species, including for example,homologues, orthologs, paralogs and nonorthologous gene displacements ofknown genes, and the interchange of genetic alterations betweenorganisms is routine and well known in the art. Accordingly, themetabolic alterations enabling biosynthesis of adipate, 6-aminocaproicacid or caprolactam described herein with reference to a particularorganism such as E. coli can be readily applied to other microorganisms,including prokaryotic and eukaryotic organisms alike. Given theteachings and guidance provided herein, those skilled in the art willknow that a metabolic alteration exemplified in one organism can beapplied equally to other organisms.

In some instances, such as when an alternative adipate, 6-aminocaproicacid or caprolactam biosynthetic pathway exists in an unrelated species,adipate, 6-aminocaproic acid or caprolactam biosynthesis can beconferred onto the host species by, for example, exogenous expression ofa paralog or paralogs from the unrelated species that catalyzes asimilar, yet non-identical metabolic reaction to replace the referencedreaction. Because certain differences among metabolic networks existbetween different organisms, those skilled in the art will understandthat the actual gene usage between different organisms may differ.However, given the teachings and guidance provided herein, those skilledin the art also will understand that the teachings and methods of theinvention can be applied to all microbial organisms using the cognatemetabolic alterations to those exemplified herein to construct amicrobial organism in a species of interest that will synthesizeadipate, 6-aminocaproic acid or caprolactam.

Host microbial organisms can be selected from, and the non-naturallyoccurring microbial organisms generated in, for example, bacteria,yeast, fungus or any of a variety of other microorganisms applicable tofermentation processes. Exemplary bacteria include species selected fromEscherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens, and Pseudomonas putida.Exemplary yeasts or fungi include species selected from Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis,Kluyveromyces marxianus, Aspergillus terreus, Aspergillus niger andPichia pastoris. For example, E. coli is a particularly useful hostorganisms since it is a well characterized microbial organism suitablefor genetic engineering. Other particularly useful host organismsinclude yeast such as Saccharomyces cerevisiae.

Methods for constructing and testing the expression levels of anon-naturally occurring adipate-, 6-aminocaproic acid- orcaprolactam-producing host can be performed, for example, by recombinantand detection methods well known in the art. Such methods can be founddescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001); and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1999).

Exogenous nucleic acid sequences involved in a pathway for production ofadipate, 6-aminocaproic acid or caprolactam can be introduced stably ortransiently into a host cell using techniques well known in the artincluding, but not limited to, conjugation, electroporation, chemicaltransformation, transduction, transfection, and ultrasoundtransformation. For exogenous expression in E. coli or other prokaryoticcells, some nucleic acid sequences in the genes or cDNAs of eukaryoticnucleic acids can encode targeting signals such as an N-terminalmitochondrial or other targeting signal, which can be removed beforetransformation into prokaryotic host cells, if desired. For example,removal of a mitochondrial leader sequence led to increased expressionin E. coli (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005). Forexogenous expression in yeast or other eukaryotic cells, genes can beexpressed in the cytosol without the addition of leader sequence, or canbe targeted to mitochondrion or other organelles, or targeted forsecretion, by the addition of a suitable targeting sequence such as amitochondrial targeting or secretion signal suitable for the host cells.Thus, it is understood that appropriate modifications to a nucleic acidsequence to remove or include a targeting sequence can be incorporatedinto an exogenous nucleic acid sequence to impart desirable properties.Furthermore, genes can be subjected to codon optimization withtechniques well known in the art to achieve optimized expression of theproteins.

An expression vector or vectors can be constructed to include one ormore adipate, 6-aminocaproic acid or caprolactam biosynthetic pathwayencoding nucleic acids as exemplified herein operably linked toexpression control sequences functional in the host organism. Expressionvectors applicable for use in the microbial host organisms of theinvention include, for example, plasmids, phage vectors, viral vectors,episomes and artificial chromosomes, including vectors and selectionsequences or markers operable for stable integration into a hostchromosome. Additionally, the expression vectors can include one or moreselectable marker genes and appropriate expression control sequences.Selectable marker genes also can be included that, for example, provideresistance to antibiotics or toxins, complement auxotrophicdeficiencies, or supply critical nutrients not in the culture media.Expression control sequences can include constitutive and induciblepromoters, transcription enhancers, transcription terminators, and thelike which are well known in the art. When two or more exogenousencoding nucleic acids are to be co-expressed, both nucleic acids can beinserted, for example, into a single expression vector or in separateexpression vectors. For single vector expression, the encoding nucleicacids can be operationally linked to one common expression controlsequence or linked to different expression control sequences, such asone inducible promoter and one constitutive promoter. The transformationof exogenous nucleic acid sequences involved in a metabolic or syntheticpathway can be confirmed using methods well known in the art. Suchmethods include, for example, nucleic acid analysis such as Northernblots or polymerase chain reaction (PCR) amplification of mRNA, orimmunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product. It is understood by thoseskilled in the art that the exogenous nucleic acid is expressed in asufficient amount to produce the desired product, and it is furtherunderstood that expression levels can be optimized to obtain sufficientexpression using methods well known in the art and as disclosed herein.

The invention additionally provides methods for producing a desiredproduct such as adipate, 6-aminocaproic acid or caprolactam. In oneembodiment, the invention provides a method for producing adipate,comprising culturing a non-naturally occurring microbial organism havingan adipate pathway, the pathway comprising at least one exogenousnucleic acid encoding an adipate pathway enzyme expressed in asufficient amount to produce adipate, under conditions and for asufficient period of time to produce adipate, the adipate pathwaycomprising succinyl-CoA:acetyl-CoA acyl transferase, 3-hydroxyacyl-CoAdehydrogenase, 3-hydroxyadipyl-CoA dehydratase,5-carboxy-2-pentenoyl-CoA reductase, and adipyl-CoA synthetase orphosphotransadipylase/adipate kinase or adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase. In another embodiment, theinvention provides a method for producing adipate, comprising culturinga non-naturally occurring microbial organism having an adipate pathway,the pathway comprising at least one exogenous nucleic acid encoding anadipate pathway enzyme expressed in a sufficient amount to produceadipate, under conditions and for a sufficient period of time to produceadipate, the adipate pathway comprising succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, 3-oxoadipate reductase,3-hydroxyadipate dehydratase, and 2-enoate reductase.

In yet another embodiment, the invention provides a method for producing6-aminocaproic acid, comprising culturing a non-naturally occurringmicrobial organism having a 6-aminocaproic acid pathway, the pathwaycomprising at least one exogenous nucleic acid encoding a 6-aminocaproicacid pathway enzyme expressed in a sufficient amount to produce6-aminocaproic acid, under conditions and for a sufficient period oftime to produce 6-aminocaproic acid, the 6-aminocaproic acid pathwaycomprising CoA-dependent aldehyde dehydrogenase and transaminase or6-aminocaproate dehydrogenase. In a further embodiment, the inventionprovides a method for producing caprolactam, comprising culturing anon-naturally occurring microbial organism having a caprolactam pathway,the pathway comprising at least one exogenous nucleic acid encoding acaprolactam pathway enzyme expressed in a sufficient amount to producecaprolactam, under conditions and for a sufficient period of time toproduce caprolactam, the caprolactam pathway comprising CoA-dependentaldehyde dehydrogenase, transaminase or 6-aminocaproate dehydrogenase,and amidohydrolase.

The invention additionally provides a method for producing adipate,comprising culturing a non-naturally occurring microbial organism havingan adipate pathway, the pathway comprising at least one exogenousnucleic acid encoding an adipate pathway enzyme expressed in asufficient amount to produce adipate, under conditions and for asufficient period of time to produce adipate, the adipate pathwaycomprising alpha-ketoadipyl-CoA synthetase,phosphotransketoadipylase/alpha-ketoadipate kinase oralpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydrogenase; 2-hydroxyadipyl-CoA dehydratase;5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferaseor adipyl-CoA hydrolase.

In still another embodiment, the invention provides a method forproducing adipate, comprising culturing a non-naturally occurringmicrobial organism having an adipate pathway, the pathway comprising atleast one exogenous nucleic acid encoding an adipate pathway enzymeexpressed in a sufficient amount to produce adipate, under conditionsand for a sufficient period of time to produce adipate, the adipatepathway comprising 2-hydroxyadipate dehydrogenase; 2-hydroxyadipyl-CoAsynthetase, phosphotranshydroxyadipylase/2-hydroxyadipate kinase or2-hydroxyadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydratase; 5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoAsynthetase, phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoAtransferase or adipyl-CoA hydrolase.

Suitable purification and/or assays to test for the production ofadipate, 6-aminocaproic acid or caprolactam can be performed using wellknown methods. Suitable replicates such as triplicate cultures can begrown for each engineered strain to be tested. For example, product andbyproduct formation in the engineered production host can be monitored.The final product and intermediates, and other organic compounds, can beanalyzed by methods such as HPLC (High Performance LiquidChromatography), GC-MS (Gas Chromatography-Mass Spectroscopy) and LC-MS(Liquid Chromatography-Mass Spectroscopy) using routine procedures wellknown in the art. The release of product in the fermentation broth canalso be tested with the culture supernatant. Byproducts and residualglucose can be quantified by HPLC using, for example, a refractive indexdetector for glucose and alcohols, and a UV detector for organic acids(Lin et al., Biotechnol. Bioeng. 90:775-779 (2005)), or other suitableassay and detection methods well known in the art. The individual enzymeactivities from the exogenous DNA sequences can also be assayed usingmethods well known in the art.

The adipate, 6-aminocaproic acid or caprolactam can be separated fromother components in the culture using a variety of methods well known inthe art. Such separation methods include, for example, extractionprocedures as well as methods that include continuous liquid-liquidextraction, pervaporation, membrane filtration, membrane separation,reverse osmosis, electrodialysis, distillation, crystallization,centrifugation, extractive filtration, ion exchange chromatography, sizeexclusion chromatography, adsorption chromatography, andultrafiltration. All of the above methods are well known in the art.

Any of the non-naturally occurring microbial organisms described hereincan be cultured to produce and/or secrete the biosynthetic products ofthe invention. For example, the adipate, 6-aminocaproic acid orcaprolactam producers can be cultured for the biosynthetic production ofadipate, 6-aminocaproic acid or caprolactam.

For the production of adipate, 6-aminocaproic acid or caprolactam, therecombinant strains are cultured in a medium with carbon source andother essential nutrients. It is highly desirable to maintain anaerobicconditions in the fermenter to reduce the cost of the overall process.Such conditions can be obtained, for example, by first sparging themedium with nitrogen and then sealing the flasks with a septum andcrimp-cap. For strains where growth is not observed anaerobically,microaerobic conditions can be applied by perforating the septum with asmall hole for limited aeration. Exemplary anaerobic conditions havebeen described previously and are well-known in the art. Exemplaryaerobic and anaerobic conditions are described, for example, in U.S.patent application Ser. No. 11/891,602, filed Aug. 10, 2007.Fermentations can be performed in a batch, fed-batch or continuousmanner, as disclosed herein.

If desired, the pH of the medium can be maintained at a desired pH, inparticular neutral pH, such as a pH of around 7 by addition of a base,such as NaOH or other bases, or acid, as needed to maintain the culturemedium at a desirable pH. The growth rate can be determined by measuringoptical density using a spectrophotometer (600 nm), and the glucoseuptake rate by monitoring carbon source depletion over time.

The growth medium can be, for example, any carbohydrate source which cansupply a source of carbon to the non-naturally occurring microorganism.Such sources include, for example, sugars such as glucose, xylose,arabinose, galactose, mannose, fructose and starch. Other sources ofcarbohydrate include, for example, renewable feedstocks and biomass.Exemplary types of biomasses that can be used as feedstocks in themethods of the invention include cellulosic biomass, hemicellulosicbiomass and lignin feedstocks or portions of feedstocks. Such biomassfeedstocks contain, for example, carbohydrate substrates useful ascarbon sources such as glucose, xylose, arabinose, galactose, mannose,fructose and starch. Given the teachings and guidance provided herein,those skilled in the art will understand that renewable feedstocks andbiomass other than those exemplified above also can be used forculturing the microbial organisms of the invention for the production ofadipate, 6-aminocaproic acid or caprolactam.

In addition to renewable feedstocks such as those exemplified above, theadipate, 6-aminocaproic acid or caprolactam microbial organisms of theinvention also can be modified for growth on syngas as its source ofcarbon. In this specific embodiment, one or more proteins or enzymes areexpressed in the adipate, 6-aminocaproic acid or caprolactam producingorganisms to provide a metabolic pathway for utilization of syngas orother gaseous carbon source.

Synthesis gas, also known as syngas or producer gas, is the majorproduct of gasification of coal and of carbonaceous materials such asbiomass materials, including agricultural crops and residues. Syngas isa mixture primarily of H₂ and CO and can be obtained from thegasification of any organic feedstock, including but not limited tocoal, coal oil, natural gas, biomass, and waste organic matter.Gasification is generally carried out under a high fuel to oxygen ratio.Although largely H₂ and CO, syngas can also include CO₂ and other gasesin smaller quantities. Thus, synthesis gas provides a cost effectivesource of gaseous carbon such as CO and, additionally, CO₂.

The Wood-Ljungdahl pathway catalyzes the conversion of CO and H₂ toacetyl-CoA and other products such as acetate. Organisms capable ofutilizing CO and syngas also generally have the capability of utilizingCO₂ and CO₂/H₂ mixtures through the same basic set of enzymes andtransformations encompassed by the Wood-Ljungdahl pathway. H₂-dependentconversion of CO₂ to acetate by microorganisms was recognized longbefore it was revealed that CO also could be used by the same organismsand that the same pathways were involved. Many acetogens have been shownto grow in the presence of CO₂ and produce compounds such as acetate aslong as hydrogen is present to supply the necessary reducing equivalents(see for example, Drake, Acetogenesis, pp. 3-60 Chapman and Hall, NewYork, (1994)). This can be summarized by the following equation:2CO₂+4H₂ +nADP+nPi→CH₃COOH+2H₂O+nATPHence, non-naturally occurring microorganisms possessing theWood-Ljungdahl pathway can utilize CO₂ and H₂ mixtures as well for theproduction of acetyl-CoA and other desired products.

The Wood-Ljungdahl pathway is well known in the art and consists of 12reactions which can be separated into two branches: (1) methyl branchand (2) carbonyl branch. The methyl branch converts syngas tomethyl-tetrahydrofolate (methyl-THF) whereas the carbonyl branchconverts methyl-THF to acetyl-CoA. The reactions in the methyl branchare catalyzed in order by the following enzymes: ferredoxinoxidoreductase, formate dehydrogenase, formyltetrahydrofolatesynthetase, methenyltetrahydrofolate cyclodehydratase,methylenetetrahydrofolate dehydrogenase and methylenetetrahydrofolatereductase. The reactions in the carbonyl branch are catalyzed in orderby the following enzymes: cobalamide corrinoid/iron-sulfur protein,methyltransferase, carbon monoxide dehydrogenase, acetyl-CoA synthase,acetyl-CoA synthase disulfide reductase and hydrogenase. Following theteachings and guidance provided herein for introducing a sufficientnumber of encoding nucleic acids to generate an adipate, 6-aminocaproicacid or caprolactam pathway, those skilled in the art will understandthat the same engineering design also can be performed with respect tointroducing at least the nucleic acids encoding the Wood-Ljungdahlenzymes absent in the host organism. Therefore, introduction of one ormore encoding nucleic acids into the microbial organisms of theinvention such that the modified organism contains the completeWood-Ljungdahl pathway will confer syngas utilization ability.

Given the teachings and guidance provided herein, those skilled in theart will understand that a non-naturally occurring microbial organismcan be produced that secretes the biosynthesized compounds of theinvention when grown on a carbon source such as a carbohydrate. Suchcompounds include, for example, adipate, 6-aminocaproic acid orcaprolactam and any of the intermediate metabolites in the adipate,6-aminocaproic acid or caprolactam pathway. All that is required is toengineer in one or more of the required enzyme activities to achievebiosynthesis of the desired compound or intermediate including, forexample, inclusion of some or all of the adipate, 6-aminocaproic acid orcaprolactam biosynthetic pathways. Accordingly, the invention provides anon-naturally occurring microbial organism that produces and/or secretesadipate, 6-aminocaproic acid or caprolactam when grown on a carbohydrateand produces and/or secretes any of the intermediate metabolites shownin the adipate, 6-aminocaproic acid or caprolactam pathway when grown ona carbohydrate. For example, the adipate producing microbial organismsof the invention can initiate synthesis from an intermediate, forexample, 3-oxoadipyl-CoA, 3-hydroxyadipyl-CoA,5-carboxy-2-pentenoyl-CoA, or adipyl-CoA (see FIG. 2), as desired. Inaddition, an adipate producing microbial organism can initiate synthesisfrom an intermediate, for example, 3-oxoadipyl-CoA, 3-oxoadipate,3-hydroxyadipate, or hexa-2-enedioate (see FIG. 3). The 6-aminocaproicacid producing microbial organism of the invention can initiatesynthesis from an intermediate, for example, adipate semialdehyde (seeFIG. 8). The caprolactam producing microbial organism of the inventioncan initiate synthesis from an intermediate, for example, adipatesemialdehyde or 6-aminocaproic acid (see FIG. 8), as desired.

The non-naturally occurring microbial organisms of the invention areconstructed using methods well known in the art as exemplified herein toexogenously express at least one nucleic acid encoding an adipate,6-aminocaproic acid or caprolactam pathway enzyme in sufficient amountsto produce adipate, 6-aminocaproic acid or caprolactam. It is understoodthat the microbial organisms of the invention are cultured underconditions sufficient to produce adipate, 6-aminocaproic acid orcaprolactam. Following the teachings and guidance provided herein, thenon-naturally occurring microbial organisms of the invention can achievebiosynthesis of adipate, 6-aminocaproic acid or caprolactam resulting inintracellular concentrations between about 0.1-200 mM or more.Generally, the intracellular concentration of adipate, 6-aminocaproicacid or caprolactam is between about 3-150 mM, particularly betweenabout 5-125 mM and more particularly between about 8-100 mM, includingabout 10 mM, 20 mM, 50 mM, 80 mM, or more. Intracellular concentrationsbetween and above each of these exemplary ranges also can be achievedfrom the non-naturally occurring microbial organisms of the invention.

In some embodiments, culture conditions include anaerobic orsubstantially anaerobic growth or maintenance conditions. Exemplaryanaerobic conditions have been described previously and are well knownin the art. Exemplary anaerobic conditions for fermentation processesare described herein and are described, for example, in U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007. Any of theseconditions can be employed with the non-naturally occurring microbialorganisms as well as other anaerobic conditions well known in the art.Under such anaerobic conditions, the adipate, 6-aminocaproic acid orcaprolactam producers can synthesize adipate, 6-aminocaproic acid orcaprolactam at intracellular concentrations of 5-10 mM or more as wellas all other concentrations exemplified herein. It is understood that,even though the above description refers to intracellularconcentrations, adipate, 6-aminocaproic acid or caprolactam producingmicrobial organisms can produce adipate, 6-aminocaproic acid orcaprolactam intracellularly and/or secrete the product into the culturemedium.

The culture conditions can include, for example, liquid cultureprocedures as well as fermentation and other large scale cultureprocedures. As described herein, particularly useful yields of thebiosynthetic products of the invention can be obtained under anaerobicor substantially anaerobic culture conditions.

As described herein, one exemplary growth condition for achievingbiosynthesis of adipate, 6-aminocaproic acid or caprolactam includesanaerobic culture or fermentation conditions. In certain embodiments,the non-naturally occurring microbial organisms of the invention can besustained, cultured or fermented under anaerobic or substantiallyanaerobic conditions. Briefly, anaerobic conditions refers to anenvironment devoid of oxygen. Substantially anaerobic conditionsinclude, for example, a culture, batch fermentation or continuousfermentation such that the dissolved oxygen concentration in the mediumremains between 0 and 10% of saturation. Substantially anaerobicconditions also includes growing or resting cells in liquid medium or onsolid agar inside a sealed chamber maintained with an atmosphere of lessthan 1% oxygen. The percent of oxygen can be maintained by, for example,sparging the culture with an N₂/CO₂ mixture or other suitable non-oxygengas or gases.

The culture conditions described herein can be scaled up and growncontinuously for manufacturing of adipate, 6-aminocaproic acid orcaprolactam. Exemplary growth procedures include, for example, fed-batchfermentation and batch separation; fed-batch fermentation and continuousseparation, or continuous fermentation and continuous separation. All ofthese processes are well known in the art. Fermentation procedures areparticularly useful for the biosynthetic production of commercialquantities of adipate, 6-aminocaproic acid or caprolactam. Generally,and as with non-continuous culture procedures, the continuous and/ornear-continuous production of adipate, 6-aminocaproic acid orcaprolactam will include culturing a non-naturally occurring adipate,6-aminocaproic acid or caprolactam producing organism of the inventionin sufficient nutrients and medium to sustain and/or nearly sustaingrowth in an exponential phase. Continuous culture under such conditionscan be include, for example, 1 day, 2, 3, 4, 5, 6 or 7 days or more.Additionally, continuous culture can include 1 week, 2, 3, 4 or 5 ormore weeks and up to several months. Alternatively, organisms of theinvention can be cultured for hours, if suitable for a particularapplication. It is to be understood that the continuous and/ornear-continuous culture conditions also can include all time intervalsin between these exemplary periods. It is further understood that thetime of culturing the microbial organism of the invention is for asufficient period of time to produce a sufficient amount of product fora desired purpose.

Fermentation procedures are well known in the art. Briefly, fermentationfor the biosynthetic production of adipate, 6-aminocaproic acid orcaprolactam can be utilized in, for example, fed-batch fermentation andbatch separation; fed-batch fermentation and continuous separation, orcontinuous fermentation and continuous separation. Examples of batch andcontinuous fermentation procedures are well known in the art.

In addition to the above fermentation procedures using the adipate,6-aminocaproic acid or caprolactam producers of the invention forcontinuous production of substantial quantities of adipate,6-aminocaproic acid or caprolactam, the adipate, 6-aminocaproic acid orcaprolactam producers also can be, for example, simultaneously subjectedto chemical synthesis procedures to convert the product to othercompounds or the product can be separated from the fermentation cultureand sequentially subjected to chemical conversion to convert the productto other compounds, if desired. As described herein, an intermediate inthe adipate pathway utilizing 3-oxoadipate, hexa-2-enedioate, can beconverted to adipate, for example, by chemical hydrogenation over aplatinum catalyst (see Example III).

To generate better producers, metabolic modeling can be utilized tooptimize growth conditions. Modeling can also be used to design geneknockouts that additionally optimize utilization of the pathway (see,for example, U.S. patent publications US 2002/0012939, US 2003/0224363,US 2004/0029149, US 2004/0072723, US 2003/0059792, US 2002/0168654 andUS 2004/0009466, and U.S. Pat. No. 7,127,379). Modeling analysis allowsreliable predictions of the effects on cell growth of shifting themetabolism towards more efficient production of adipate, 6-aminocaproicacid or caprolactam.

One computational method for identifying and designing metabolicalterations favoring biosynthesis of a desired product is the OptKnockcomputational framework, Burgard et al., Biotechnol. Bioeng. 84:647-657(2003). OptKnock is a metabolic modeling and simulation program thatsuggests gene deletion strategies that result in genetically stablemicroorganisms which overproduce the target product. Specifically, theframework examines the complete metabolic and/or biochemical network ofa microorganism in order to suggest genetic manipulations that force thedesired biochemical to become an obligatory byproduct of cell growth. Bycoupling biochemical production with cell growth through strategicallyplaced gene deletions or other functional gene disruption, the growthselection pressures imposed on the engineered strains after long periodsof time in a bioreactor lead to improvements in performance as a resultof the compulsory growth-coupled biochemical production. Lastly, whengene deletions are constructed there is a negligible possibility of thedesigned strains reverting to their wild-type states because the genesselected by OptKnock are to be completely removed from the genome.Therefore, this computational methodology can be used to either identifyalternative pathways that lead to biosynthesis of a desired product orused in connection with the non-naturally occurring microbial organismsfor further optimization of biosynthesis of a desired product.

Briefly, OptKnock is a term used herein to refer to a computationalmethod and system for modeling cellular metabolism. The OptKnock programrelates to a framework of models and methods that incorporate particularconstraints into flux balance analysis (FBA) models. These constraintsinclude, for example, qualitative kinetic information, qualitativeregulatory information, and/or DNA microarray experimental data.OptKnock also computes solutions to various metabolic problems by, forexample, tightening the flux boundaries derived through flux balancemodels and subsequently probing the performance limits of metabolicnetworks in the presence of gene additions or deletions. OptKnockcomputational framework allows the construction of model formulationsthat enable an effective query of the performance limits of metabolicnetworks and provides methods for solving the resulting mixed-integerlinear programming problems. The metabolic modeling and simulationmethods referred to herein as OptKnock are described in, for example,U.S. publication 2002/0168654, filed Jan. 10, 2002, in InternationalPatent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S. patentapplication Ser. No. 11/891,602, filed Aug. 10, 2007.

Another computational method for identifying and designing metabolicalterations favoring biosynthetic production of a product is a metabolicmodeling and simulation system termed SimPheny®. This computationalmethod and system is described in, for example, U.S. publication2003/0233218, filed Jun. 14, 2002, and in International PatentApplication No. PCT/US03/18838, filed Jun. 13, 2003. SimPheny® is acomputational system that can be used to produce a network model insilico and to simulate the flux of mass, energy or charge through thechemical reactions of a biological system to define a solution spacethat contains any and all possible functionalities of the chemicalreactions in the system, thereby determining a range of allowedactivities for the biological system. This approach is referred to asconstraints-based modeling because the solution space is defined byconstraints such as the known stoichiometry of the included reactions aswell as reaction thermodynamic and capacity constraints associated withmaximum fluxes through reactions. The space defined by these constraintscan be interrogated to determine the phenotypic capabilities andbehavior of the biological system or of its biochemical components.

These computational approaches are consistent with biological realitiesbecause biological systems are flexible and can reach the same result inmany different ways. Biological systems are designed throughevolutionary mechanisms that have been restricted by fundamentalconstraints that all living systems must face. Therefore,constraints-based modeling strategy embraces these general realities.Further, the ability to continuously impose further restrictions on anetwork model via the tightening of constraints results in a reductionin the size of the solution space, thereby enhancing the precision withwhich physiological performance or phenotype can be predicted.

Given the teachings and guidance provided herein, those skilled in theart will be able to apply various computational frameworks for metabolicmodeling and simulation to design and implement biosynthesis of adesired compound in host microbial organisms. Such metabolic modelingand simulation methods include, for example, the computational systemsexemplified above as SimPheny® and OptKnock. For illustration of theinvention, some methods are described herein with reference to theOptKnock computation framework for modeling and simulation. Thoseskilled in the art will know how to apply the identification, design andimplementation of the metabolic alterations using OptKnock to any ofsuch other metabolic modeling and simulation computational frameworksand methods well known in the art.

The methods described above will provide one set of metabolic reactionsto disrupt. Elimination of each reaction within the set or metabolicmodification can result in a desired product as an obligatory productduring the growth phase of the organism. Because the reactions areknown, a solution to the bilevel OptKnock problem also will provide theassociated gene or genes encoding one or more enzymes that catalyze eachreaction within the set of reactions. Identification of a set ofreactions and their corresponding genes encoding the enzymesparticipating in each reaction is generally an automated process,accomplished through correlation of the reactions with a reactiondatabase having a relationship between enzymes and encoding genes.

Once identified, the set of reactions that are to be disrupted in orderto achieve production of a desired product are implemented in the targetcell or organism by functional disruption of at least one gene encodingeach metabolic reaction within the set. One particularly useful means toachieve functional disruption of the reaction set is by deletion of eachencoding gene. However, in some instances, it can be beneficial todisrupt the reaction by other genetic aberrations including, forexample, mutation, deletion of regulatory regions such as promoters orcis binding sites for regulatory factors, or by truncation of the codingsequence at any of a number of locations. These latter aberrations,resulting in less than total deletion of the gene set can be useful, forexample, when rapid assessments of the coupling of a product are desiredor when genetic reversion is less likely to occur.

To identify additional productive solutions to the above describedbilevel OptKnock problem which lead to further sets of reactions todisrupt or metabolic modifications that can result in the biosynthesis,including growth-coupled biosynthesis of a desired product, anoptimization method, termed integer cuts, can be implemented. Thismethod proceeds by iteratively solving the OptKnock problem exemplifiedabove with the incorporation of an additional constraint referred to asan integer cut at each iteration. Integer cut constraints effectivelyprevent the solution procedure from choosing the exact same set ofreactions identified in any previous iteration that obligatorily couplesproduct biosynthesis to growth. For example, if a previously identifiedgrowth-coupled metabolic modification specifies reactions 1, 2, and 3for disruption, then the following constraint prevents the samereactions from being simultaneously considered in subsequent solutions.The integer cut method is well known in the art and can be founddescribed in, for example, Burgard et al., Biotechnol. Prog. 17:791-797(2001). As with all methods described herein with reference to their usein combination with the OptKnock computational framework for metabolicmodeling and simulation, the integer cut method of reducing redundancyin iterative computational analysis also can be applied with othercomputational frameworks well known in the art including, for example,SimPheny®.

The methods exemplified herein allow the construction of cells andorganisms that biosynthetically produce a desired product, including theobligatory coupling of production of a target biochemical product togrowth of the cell or organism engineered to harbor the identifiedgenetic alterations. Therefore, the computational methods describedherein allow the identification and implementation of metabolicmodifications that are identified by an in silico method selected fromOptKnock or SimPheny®. The set of metabolic modifications can include,for example, addition of one or more biosynthetic pathway enzymes and/orfunctional disruption of one or more metabolic reactions including, forexample, disruption by gene deletion.

As discussed above, the OptKnock methodology was developed on thepremise that mutant microbial networks can be evolved towards theircomputationally predicted maximum-growth phenotypes when subjected tolong periods of growth selection. In other words, the approach leveragesan organism's ability to self-optimize under selective pressures. TheOptKnock framework allows for the exhaustive enumeration of genedeletion combinations that force a coupling between biochemicalproduction and cell growth based on network stoichiometry. Theidentification of optimal gene/reaction knockouts requires the solutionof a bilevel optimization problem that chooses the set of activereactions such that an optimal growth solution for the resulting networkoverproduces the biochemical of interest (Burgard et al., Biotechnol.Bioeng. 84:647-657 (2003)).

An in silico stoichiometric model of E. coli metabolism can be employedto identify essential genes for metabolic pathways as exemplifiedpreviously and described in, for example, U.S. patent publications US2002/0012939, US 2003/0224363, US 2004/0029149, US 2004/0072723, US2003/0059792, US 2002/0168654 and US 2004/0009466, and in U.S. Pat. No.7,127,379. As disclosed herein, the OptKnock mathematical framework canbe applied to pinpoint gene deletions leading to the growth-coupledproduction of a desired product. Further, the solution of the bilevelOptKnock problem provides only one set of deletions. To enumerate allmeaningful solutions, that is, all sets of knockouts leading togrowth-coupled production formation, an optimization technique, termedinteger cuts, can be implemented. This entails iteratively solving theOptKnock problem with the incorporation of an additional constraintreferred to as an integer cut at each iteration, as discussed above.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoprovided within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

Example I Reverse Adipate Degradation Pathway

This example describes an exemplary adipate synthesis pathway via areverse adipate degradation pathway.

Organisms such as Penicillium chrysogenum have the ability to naturallydegrade adipate (Thykaer et al., Metab. Eng. 4:151-158. (2002)). Themechanism is similar to the oxidation of fatty acids (see FIG. 1). Thefirst step in adipate degradation is an ATP-dependent reaction thatactivates adipate with CoA. The second reaction is catalyzed by adehydrogenase that forms 5-carboxy-2-pentenoyl-CoA from adipyl-CoA.During peroxisomal adipate degradation, the dehydrogenase enzymecontains FAD, which accepts the electrons and then transfers themdirectly to oxygen. A catalase enzyme dissipates the H₂O₂ formed by thereduction of oxygen. In mitochondrial fatty acid oxidation, the FAD fromthe dehydrogenase transfers electrons directly to the electron transportchain. A multi-functional fatty acid oxidation protein in eukaryotessuch as S. cerevisiae and P. chrysogenum carries out the followinghydratase and dehydrogenase steps. The final step is an acyl transferasethat splits 3-oxoadipyl CoA into acetyl-CoA and succinyl-CoA.

A highly efficient pathway for the production of adipate is achievedthrough genetically altering a microorganism such that similar enzymaticreactions are employed for adipate synthesis from succinyl-CoA andacetyl-CoA (see FIG. 2). Successful implementation of this entailsexpressing the appropriate genes, tailoring their expression, andaltering culture conditions so that high acetyl-CoA, succinyl-CoA,and/or redox (for example, NADH/NAD+) ratios will drive the metabolicflux through this pathway in the direction of adipate synthesis ratherthan degradation. Strong parallels to butyrate formation in Clostridia(Kanehisa and Goto, Nucl. Acids Res. 28:27-30 (2000)) support that eachstep in the adipate synthesis pathway is thermodynamically feasible withreaction directionality governed by the concentrations of theparticipating metabolites. The final step, which forms adipate fromadipyl-CoA, can take place either via a synthetase,phosphotransadipylase/kinase, transferase, or hydrolase mechanism.

The maximum theoretical yields of adipate using this pathway werecalculated both in the presence and absence of an external electronacceptor such as oxygen. These calculations show that the pathway canefficiently transform glucose into adipate and CO₂ under anaerobicconditions with a 92% molar yield (Table I). The production of adipateusing this pathway does not require the uptake of oxygen as NAD+ can beregenerated in the two hydrogenase steps that form 3-hydroxyadipyl-CoAand adipyl-CoA (see FIG. 2). Further, the pathway is favorableenergetically as up to 1.55 moles of ATP are formed per mole of glucoseconsumed at the maximum theoretical yield of adipate assuming either asynthetase, phosphotransadipylase/kinase, or transferase mechanism forthe final conversion step. The ATP yield can be further improved to 2.47moles of ATP produced per mole of glucose if phosphoenolpyruvatecarboxykinase (PPCK) is assumed to function in the ATP-generatingdirection towards oxaloacetate formation. Maximum ATP yield calculationswere then performed assuming that the adipyl-CoA to adipatetransformation is a hydrolysis step. This reduces the maximum ATP yieldsat maximum adipate production to 0.85 and 1.77 mole ATP per mole glucoseconsumed if PPCK is assumed irreversible and reversible, respectively.Nevertheless, these ATP yields are sufficient for cell growth,maintenance, and production.

TABLE 1 The maximum theoretical yields of adipate and the associated ATPyields per mole of glucose using the reverse degradation pathwayassuming the final step in the pathway is a synthetase,phosphotransadipylase/kinase, or transferase. Aerobic Anaerobic AdipateYield 0.92 0.92 Max ATP yield @ max adipate yield 1.55 1.55 Max ATPyield @ max adipate yield 2.47 2.47 PPCK assumed

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of adipate, one or moreexogenous DNA sequence(s) are expressed in a suitable hostmicroorganism. In addition, the microorganisms can have endogenousgene(s) functionally deleted. These modifications allow the productionof adipate using renewable feedstock.

Below is described a number of biochemically characterized candidategenes that encode enzymes that catalyze each step of the reverse adipatedegradation pathway in a production host. Although described using E.coli as a host organism to engineer the pathway, essentially anysuitable host organism can be used. Specifically listed are genes thatare native to E. coli as well as genes in other organisms that can beapplied to catalyze the appropriate transformations when properly clonedand expressed.

Referring to FIG. 2, step 1 involves succinyl CoA:acetyl CoA acyltransferase (β-ketothiolase). The first step in the pathway combinesacetyl-CoA and succinyl-CoA to form 3-oxoadipyl-CoA. The gene productsencoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J.Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera etal., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), paaE inPseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol.188:117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiol.153:357-365 (2007)) catalyze the conversion of 3-oxoadipyl-CoA intosuccinyl-CoA and acetyl-CoA during the degradation of aromatic compoundssuch as phenylacetate or styrene. Since β-ketothiolase enzymes catalyzereversible transformations, these enzymes can be employed for the firststep in adipate synthesis shown in FIG. 2. For example, the ketothiolasephaA from R. eutropha combines two molecules of acetyl-CoA to formacetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)).Similarly, a β-keto thiolase (bktB) has been reported to catalyze thecondensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA(Slater et al., J. Bacteriol. 180: 1979-1987 (1998)) in R. eutropha. Theprotein sequences for the above-mentioned gene products are well knownin the art and can be accessed in the public databases such as GenBankusing the following accession numbers.

Gene name GenBank Accession # Organism paaJ NP_415915.1 Escherichia colipcaF AAL02407 Pseudomonas knackmussii (B13) phaD AAC24332.1 Pseudomonasputida paaE ABF82237.1 Pseudomonas fluorescens

These exemplary sequences can be used to identify homologue proteins inGenBank or other databases through sequence similarity searches (forexample, BLASTp). The resulting homologue proteins and theircorresponding gene sequences provide additional exogenous DNA sequencesfor transformation into E. coli or other suitable host microorganisms togenerate production hosts.

For example, orthologs of paaJ from Escherichia coli K12 can be foundusing the following GenBank accession numbers:

YP_001335140.1 Klebsiella pneumoniae YP_001479310.1 Serratiaproteamaculans AAC24332.1 Pseudomonas putida

Example orthologs of pcaF from Pseudomonas knackmussii can be foundusing the following GenBank accession numbers:

AAD22035.1 Streptomyces sp. 2065 AAN67000.1 Pseudomonas putidaABJ15177.1 Pseudomonas aeruginosa

Additional native candidate genes for the ketothiolase step includeatoB, which can catalyze the reversible condensation of 2 acetyl-CoAmolecules (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)), andits homolog yqeF. Non-native gene candidates include phaA (Sato et al.,supra, 2007) and bktB (Slater et al., J. Bacteriol. 180:1979-1987(1998)) from R. eutropha, and the two ketothiolases, thiA and thiB, fromClostridium acetobutylicum (Winzer et al., J. Mol. Microbiol.Biotechnol. 2:531-541 (2000)). The protein sequences for each of theseexemplary gene products can be found using the following GenBankaccession numbers:

atoB NP_416728.1 Escherichia coli yqeF NP_417321.2 Escherichia coli phaAYP_725941 Ralstonia eutropha bktB AAC38322.1 Ralstonia eutropha thiANP_349476.1 Clostridium acetobutylicum thiB NP_149242.1 Clostridiumacetobutylicum

Referring to FIG. 2, step 2 involves 3-hydroxyacyl-CoA dehydrogenase.The second step in the pathway involves the reduction of 3-oxoadipyl-CoAto 3-hydroxyadipyl-CoA. The gene products encoded by phaC in Pseudomonasputida U (Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424(1998)) and paaC in Pseudomonas fluorescens ST (Di Gennaro et al., Arch.Microbiol. 188:117-125 (2007)) catalyze the reverse reaction, that is,the oxidation of 3-hydroxyadipyl-CoA to form 3-oxoadipyl-CoA, during thecatabolism of phenylacetate or styrene. The reactions catalyzed by suchdehydrogenases are reversible and accordingly these genes representcandidates to carry out the second step of adipate synthesis as shown inFIG. 2. A similar transformation is also carried out by the gene productof hbd in Clostridium acetobutylicum (Atsumi et al., Metab. Eng. (epubSep. 14, 2007); Boynton et al., J. Bacteriol. 178:3015-3024 (1996)).This enzyme converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Lastly,given the proximity in E. coli of paaH to other genes in thephenylacetate degradation operon (Nogales et al., Microbiol. 153:357-365(2007)) and the fact that paaH mutants cannot grow on phenylacetate(Ismail et al., Eur. J. Biochem. 270:3047-3054 (2003)), it is expectedthat the E. coli paaH gene encodes a 3-hydroxyacyl-CoA dehydrogenase.The protein sequences for each of these exemplary gene products can befound using the following GenBank accession numbers:

paaH NP_415913.1 Escherichia coli phaC NP_745425.1 Pseudomonas putidapaaC ABF82235.1 Pseudomonas fluorescens hbd NP_349314.1 Clostridiumacetobutylicum

Referring to FIG. 2, step 3 involves 3-hydroxyadipyl-CoA dehydratase.The gene product of crt from C. acetobutylicum catalyzes the dehydrationof 3-hydroxybutyryl-CoA to crotonyl-CoA (see FIG. 2) (Atsumi et al.,supra, 2007; Boynton et al., J. Bacteriol. 178:3015-3024 (1996)).Homologs of this gene are strong candidates for carrying out the thirdstep in the adipate synthesis pathway exemplified in FIG. 2. Inaddition, genes known to catalyze the hydroxylation of double bonds inenoyl-CoA compounds represent additional candidates given thereversibility of such enzymatic transformations. For example, theenoyl-CoA hydratases, phaA and phaB, of P. putida are believed to carryout the hydroxylation of double bonds during phenylacetate catabolism(Olivera et al., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)) andthus represent additional candidates for incorporation into E. coli. Thedeletion of these genes precludes phenylacetate degradation in P.putida. The paaA and paaB from P. fluorescens catalyze analogoustransformations (Olivera et al., supra, 1998). Lastly, a number ofEscherichia coli genes have been shown to demonstrate enoyl-CoAhydratase functionality including maoC (Park and Lee, J. Bacteriol.185:5391-5397 (2003)), paaF (Ismail et al., Eur. J. Biochem.270:3047-3054 (2003); Park and Lee, Biotechnol. Bioeng. 86:681-686(2004); Park and Lee, Appl. Biochem. Biotechnol. 113-116:335-346(2004)), and paaG (Ismail et al., supra, 2003; Park and Lee, supra,2004; Park and Lee, supra, 2004). The protein sequences for each ofthese exemplary gene products can be found using the following GenBankaccession numbers:

maoC NP_415905.1 Escherichia coli paaF NP_415911.1 Escherichia coli paaGNP_415912.1 Escherichia coli crt NP_349318.1 Clostridium acetobutylicumpaaA NP_745427.1 Pseudomonas putida paaB NP_745426.1 Pseudomonas putidaphaA ABF82233.1 Pseudomonas fluorescens phaB ABF82234.1 Pseudomonasfluorescens

Alternatively, β-oxidation genes are candidates for the first threesteps in adipate synthesis. Candidate genes for the proposed adipatesynthesis pathway also include the native fatty acid oxidation genes ofE. coli and their homologs in other organisms. The E. coli genes fadAand fadB encode a multienzyme complex that exhibits ketoacyl-CoAthiolase, 3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydrataseactivities (Yang et al., Biochem. 30:6788-6795 (1991); Yang et al.,Biol. Chem. 265:10424-10429 (1990); Yang et al., Biol. Chem. 266:16255(1991); Nakahigashi and Inokuchi, Nucl. Acids Res. 18: 4937 (1990)).These activities are mechanistically similar to the first threetransformations shown in FIG. 2. The fadI and fadJ genes encode similarfunctions and are naturally expressed only anaerobically (Campbell etal., Mol. Microbiol. 47:793-805 (2003)). These gene products naturallyoperate to degrade short, medium, and long chain fatty-acyl-CoAcompounds to acetyl-CoA, rather than to convert succinyl-CoA andacetyl-CoA into 5-carboxy-2-pentenoyl-CoA as proposed in FIG. 2.However, it is well known that the ketoacyl-CoA thiolase,3-hydroxyacyl-CoA dehydrogenase, and enoyl-CoA hydratase enzymescatalyze reversible transformations. Furthermore, directed evolution andrelated approaches can be applied to tailor the substrate specificitiesof the native β-oxidation machinery of E. coli. Thus these enzymes orhomologues thereof can be applied for adipate production. If the nativegenes operate to degrade adipate or its precursors in vivo, theappropriate genetic modifications are made to attenuate or eliminatethese functions. However, it may not be necessary since a method forproducing poly[(R)-3-hydroxybutyrate] in E. coli that involvesactivating fadB, by knocking out a negative regulator, fadR, andco-expressing a non-native ketothiolase, phaA from Ralstonia eutropha,has been described (Sato et al., J. Biosci. Bioeng. 103:38-44 (2007)).This work clearly demonstrated that a β-oxidation enzyme, in particularthe gene product of fadB which encodes both 3-hydroxyacyl-CoAdehydrogenase and enoyl-CoA hydratase activities, can function as partof a pathway to produce longer chain molecules from acetyl-CoAprecursors. The protein sequences for each of these exemplary geneproducts can be found using the following GenBank accession numbers:

fadA YP_026272.1 Escherichia coli fadB NP_418288.1 Escherichia coli fadINP_416844.1 Escherichia coli fadJ NP_416843.1 Escherichia coli fadRNP_415705.1 Escherichia coli

Referring to FIG. 2, step 4 involves 5-carboxy-2-pentenoyl-CoAreductase. Whereas the ketothiolase, dehydrogenase, and enoyl-CoAhydratase steps are generally reversible, the enoyl-CoA reductase stepis almost always oxidative and irreversible under physiologicalconditions (Hoffmeister et al., J. Biol. Chem. 280:4329-4338 (2005)).FadE catalyzes this likely irreversible transformation in E. coli(Campbell and Cronan, J. Bacteriol. 184:3759-3764 (2002)). The pathwayrequires an enzyme that can reduce a 2-enoyl-CoA intermediate, not onesuch as FadE that will only oxidize an acyl-CoA to a 2-enoyl-CoAcompound. Furthermore, although it has been suggested that E. colinaturally possesses enzymes for enoyl-CoA reduction (Mizugaki et al., J.Biochem. 92:1649-1654 (1982); Nishimaki et al., J. Biochem. 95:1315-1321(1984)), no E. coli gene possessing this function has been biochemicallycharacterized.

One candidate gene for the enoyl-CoA reductase step is the gene productof bcd from C. acetobutylicum (Atsumi et al., supra, 2007; Boynton etal., J. Bacteriol. 178:3015-3024 (1996)), which naturally catalyzes thereduction of crotonyl-CoA to butyryl-CoA, a reaction similar inmechanism to the desired reduction of 5-carboxy-2-pentenoyl-CoA toadipyl-CoA in the adipate synthesis pathway. Activity of this enzyme canbe enhanced by expressing bcd in conjunction with expression of the C.acetobutylicum etfAB genes, which encode an electron transferflavoprotein. An additional candidate for the enoyl-CoA reductase stepis the mitochondrial enoyl-CoA reductase from E. gracilis (Hoffmeisteret al., J. Biol. Chem. 280:4329-4338 (2005)). A construct derived fromthis sequence following the removal of its mitochondrial targetingleader sequence was cloned in E. coli, resulting in an active enzyme(Hoffmeister et al., supra, 2005). This approach is well known to thoseskilled in the art of expressing eukarytotic genes, particularly thosewith leader sequences that may target the gene product to a specificintracellular compartment, in prokaryotic organisms. A close homolog ofthis gene, TDE0597, from the prokaryote Treponema denticola represents athird enoyl-CoA reductase which has been cloned and expressed in E. coli(Tucci and Martin, FEBS Lett. 581:1561-1566 (2007)). The proteinsequences for each of these exemplary gene products can be found usingthe following GenBank accession numbers:

bcd NP_349317.1 Clostridium acetobutylicum etfA NP_349315.1 Clostridiumacetobutylicum etfB NP_349316.1 Clostridium acetobutylicum TER Q5EU90.1Euglena gracilis TDE0597 NP_971211.1 Treponema denticola

Referring to FIG. 2, step 5 involves adipyl-CoA synthetase (alsoreferred to as adipate-CoA ligase), phosphotransadipylase/adipatekinase, adipyl-CoA:acetyl-CoA transferase, or adipyl-CoA hydrolase. Froman energetic standpoint, it is desirable for the final step in theadipate synthesis pathway to be catalyzed by an enzyme or enzyme pairthat can conserve the ATP equivalent stored in the thioester bond ofadipyl-CoA. The product of the sucC and sucD genes of E. coli, orhomologs thereof, can potentially catalyze the final transformationshown in FIG. 2 should they exhibit activity on adipyl-CoA. The sucCDgenes naturally form a succinyl-CoA synthetase complex that catalyzesthe formation of succinyl-CoA from succinate with the concaminantconsumption of one ATP, a reaction which is reversible in vivo (Buck etal., Biochem. 24:6245-6252 (1985)). Given the structural similaritybetween succinate and adipate, that is, both are straight chaindicarboxylic acids, it is reasonable to expect some activity of thesucCD enzyme on adipyl-CoA. An enzyme exhibiting adipyl-CoA ligaseactivity can equivalently carry out the ATP-generating production ofadipate from adipyl-CoA, here using AMP and PPi as cofactors, whenoperating in the opposite physiological direction as depicted in FIG. 1.Exemplary CoA-ligases include the rat dicarboxylate-CoA ligase for whichthe sequence is yet uncharacterized (Vamecq et al., Biochem. J.230:683-693 (1985)), either of the two characterized phenylacetate-CoAligases from P. chrysogenum (Lamas-Maceiras et al., Biochem. J. 395,147-155 (2005); Wang et al., Biochem. Biophy. Res. Commun. 360:453-458(2007)), the phenylacetate-CoA ligase from Pseudomonas putida(Martinez-Blanco et al., J. Biol. Chem. 265:7084-7090 (1990)), and the6-carboxyhexanoate-CoA ligase from Bacillus subtilis (Bower et al., J.Bacteriol. 178:4122-4130 (1996)). The protein sequences for each ofthese exemplary gene products can be found using the following GenBankaccession numbers:

sucC NP_415256.1 Escherichia coli sucD AAC73823.1 Escherichia coli

Another option, using phosphotransadipylase/adipate kinase, is catalyzedby the gene products of buk1, buk2, and ptb from C. acetobutylicum(Walter et al., Gene 134:107-111 (1993); Huang et al., J. Mol.Microbiol. Biotechnol. 2:33-38 (2000)), or homologs thereof. The ptbgene encodes an enzyme that can convert butyryl-CoA intobutyryl-phosphate, which is then converted to butyrate via either of thebuk gene products with the concomitant generation of ATP. The analogousset of transformations, that is, conversion of adipyl-CoA toadipyl-phosphate followed by conversion of adipyl-phosphate to adipate,can be carried out by the buk1, buk2, and ptb gene products. The proteinsequences for each of these exemplary gene products can be found usingthe following GenBank accession numbers:

ptb NP_349676 Clostridium acetobutylicum buk1 NP_349675 Clostridiumacetobutylicum buk2 Q97II1 Clostridium acetobutylicum

Alternatively, an acetyltransferase capable of transferring the CoAgroup from adipyl-CoA to acetate can be applied. Similar transformationsare catalyzed by the gene products of cat1, cat2, and cat3 ofClostridium kluyveri which have been shown to exhibit succinyl-CoA,4-hydroxybutyryl-CoA, and butyryl-CoA acetyltransferase activity,respectively (Sohling and Gottschalk, J. Bacteriol. 178:871-880 (1996);Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). Theprotein sequences for each of these exemplary gene products can be foundusing the following GenBank accession numbers:

cat1 P38946.1 Clostridium kluyveri cat2 P38942.2 Clostridium kluyvericat3 EDK35586.1 Clostridium kluyveri

Finally, though not as desirable from an energetic standpoint, theconversion of adipyl-CoA to adipate can also be carried out by anacyl-CoA hydrolase or equivalently a thioesterase. The top E. coli genecandidate is tesB (Naggert et al., J. Biol. Chem. 266:11044-11050(1991)), which shows high similarity to the human acot8, which is adicarboxylic acid acetyltransferase with activity on adipyl-CoA (Westinet al., J. Biol. Chem. 280:38125-38132 (2005)). This activity has alsobeen characterized in the rat liver (Deana, Biochem. Int. 26:767-773(1992)). The protein sequences for each of these exemplary gene productscan be found using the following GenBank accession numbers:

tesB NP_414986 Escherichia coli acot8 CAA15502 Homo sapiens acot8NP_570112 Rattus norvegicus

Other native candidate genes include tesA (Bonner and Bloch, J. Biol.Chem. 247:3123-3133 (1972)), ybgC (Kuznetsova et al., FEMS Microbiol.Rev. 29:263-279 (2005); Zhuang et al., FEBS Lett. 516:161-163 (2002)),paaI (Song et al., J. Biol. Chem. 281:11028-11038 (2006)), and ybdB(Leduc et al., J. Bacteriol. 189:7112-7126 (2007)). The proteinsequences for each of these exemplary gene products can be found usingthe following GenBank accession numbers:

tesA NP_415027 Escherichia coli ybgC NP_415264 Escherichia coli paaINP_415914 Escherichia coli ybdB NP_415129 Escherichia coli

The above description provides an exemplary adipate synthesis pathway byway of a reverse adipate degradation pathway.

Example II Preparation of an Adipate Producing Microbial Organism Havinga Reverse Degradation Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using the reverse degradation pathway.

Escherichia coli is used as a target organism to engineer a reverseadipate degradation pathway as shown in FIG. 2. E. coli provides a goodhost for generating a non-naturally occurring microorganism capable ofproducing adipate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleicacids encoding the enzymes utilized in the reverse degradation pathwayare expressed in E. coli using well known molecular biology techniques(see, for example, Sambrook, supra, 2001; Ausubel supra, 1999). Inparticular, the paaJ (NP_415915.1), paaH (NP_415913.1), and maoC(NP_415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoAdehydratase activities, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Inaddition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1), andsucCD (NP_415256.1 and AAC73823.1) genes encoding5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetaseactivities, respectively, are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. The two sets ofplasmids are transformed into E. coli strain MG1655 to express theproteins and enzymes required for adipate synthesis via the reversedegradation pathway.

The resulting genetically engineered organism is cultured inglucose-containing medium following procedures well known in the art(see, for example, Sambrook et al., supra, 2001). The expression ofreverse degradation pathway genes is corroborated using methods wellknown in the art for determining polypeptide expression or enzymaticactivity, including for example, Northern blots, PCR amplification ofmRNA, immunoblotting, and the like. Enzymatic activities of theexpressed enzymes are confirmed using assays specific for the individualactivities. The ability of the engineered E. coli strain to produceadipate is confirmed using HPLC, gas chromatography-mass spectrometry(GCMS) and/or liquid chromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of adipate. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of adipate.Adaptive evolution also can be used to generate better producers of, forexample, the acetyl-CoA and succinyl-CoA intermediates or the adipateproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the adipate producer to further increaseproduction.

For large-scale production of adipate, the above reverse degradationpathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing the culture vessel, forexample, flasks can be sealed with a septum and crimp-cap. Microaerobicconditions also can be utilized by providing a small hole in the septumfor limited aeration. The pH of the medium is maintained at a pH ofaround 7 by addition of an acid, such as H₂SO₄. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm) and the glucose uptake rate by monitoring carbon source depletionover time. Byproducts such as undesirable alcohols, organic acids, andresidual glucose can be quantified by HPLC (Shimadzu, Columbia Md.), forexample, using an Aminex® series of HPLC columns (for example, HPX-87series) (BioRad, Hercules Calif.), using a refractive index detector forglucose and alcohols, and a UV detector for organic acids (Lin et al.,Biotechnol. Bioeng. 775-779 (2005)).

This example describes the preparation of an adipate producing microbialorganism using a reverse degradation pathway.

Example III Adipate Synthesis Through 3-Oxoadipate

This example describes an exemplary adipate synthesis pathway through3-oxoadipate.

An additional pathway from that described in Examples I and II that usesacetyl-CoA and succinyl-CoA as precursors for adipate formation andpasses through the metabolic intermediate, 3-oxoadipate, is shown inFIG. 3. The initial two transformations in this pathway are the twoterminal steps of the degradation pathway for aromatic andcholoroaromatic compounds operating in the reverse direction (Kaschabeket al., J. Bacteriol. 184:207-215 (2002); Nogales et al., Microbiol.153:357-365 (2007); Ismail et al., Eur. J. Biochem. 270:3047-3054(2003)). Specifically, the first step forms 3-oxoadipyl CoA by thecondensation of succinyl- and acetyl-CoA. The second step forms3-oxoadipate and is reported to be reversible in Pseudomonas sp. StrainB13 (Kaschabek et al., J. Bacteriol. 184:207-215 (2002)).

The subsequent steps involve reduction of 3-oxoadipate to3-hydroxyadipate (conversion of a keto group to hydroxyl group),dehydration of 3-hydroxyadipate to yield hexa-2-enedioate, and reductionof hexa-2-enedioate to form adipate. These steps of the pathway areanalogous to the conversion of oxaloacetate into succinate via thereductive TCA cycle (see FIG. 4). This supports the steps in the pathwaybeing thermodynamically favorable subject to the presence of appropriatemetabolite concentrations. The final reduction step can be carried outeither biochemically or by employing a chemical catalyst to converthexa-2-enedioate into adipate. Chemical hydrogenation can be performedusing Pt catalyst on activated carbon as has been described in (Niu etal., Biotechnol. Prog. 18:201-211 (2002)).

The maximum theoretical yield of adipate using this pathway is 0.92 moleper mole glucose consumed, and oxygen is not required for attainingthese yields (see Table 2). The associated energetics are identical tothose of the reverse adipate pathway. Theoretically, ATP formation of upto 1.55 moles is observed per mole of glucose utilized through thispathway. The ATP yield improves to approximately 2.47 moles ifphosphoenolpyruvate kinase (PPCK) is assumed to operate in the directionof ATP generation. Interestingly, the product yield can be increasedfurther to 1 mole adipate per mole of glucose consumed if chemicalhydrogenation is used for the last step and a 100% efficiency ofcatalysis is assumed. In this scenario, up to 1.95 moles of ATP areformed theoretically without assuming the reverse functionality of PPCK.

TABLE 2 The maximum theoretical yields of adipate and the associated ATPyields per mole of glucose using the 3-oxoadipate pathway. Final stepFinal step enzymatic chemical hydrogenation Aerobic Anaerobic AerobicAnaerobic Adipate Yield 0.92 0.92 1.00 1.00 Max ATP yield @ 1.55 1.551.95 1.95 max adipate yield

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of adipate, one or moreexogenous DNA sequence(s) can be expressed in a host microorganism. Inaddition, the host microorganism can have endogenous gene(s)functionally deleted. These modifications allow the production ofadipate using renewable feedstock.

Described below are a number of biochemically characterized candidategenes capable of encoding enzymes that catalyze each step of the3-oxoadipate pathway for adipate synthesis. Although this method isdescribed for E. coli, one skilled in the art can apply these teachingsto any other suitable host organism. Specifically, listed below aregenes that are native to E. coli as well as genes in other organismsthat can be applied to catalyze the appropriate transformations whenproperly cloned and expressed.

Referring to FIG. 3, step 1 involves succinyl CoA:acetyl CoA acyltransferase (β-ketothiolase). The first step in the pathway combinesacetyl-CoA and succinyl-CoA to form 3-oxoadipyl-CoA. The gene productsencoded by pcaF in Pseudomonas strain B13 (Kaschabek et al., J.Bacteriol. 184:207-215 (2002)), phaD in Pseudomonas putida U (Olivera etal., Proc. Natl. Acad. Sci. USA 95:6419-6424 (1998)), paaE inPseudomonas fluorescens ST (Di Gennaro et al., Arch. Microbiol.188:117-125 (2007)), and paaJ from E. coli (Nogales et al., Microbiol.153:357-365 (2007)) catalyze the conversion of 3-oxoadipyl-CoA intosuccinyl-CoA and acetyl-CoA during the degradation of aromatic compoundssuch as phenylacetate or styrene. Since β-ketothiolase enzymes catalyzereversible transformations, these enzymes can be employed for the firststep in adipate synthesis shown in FIG. 3. For example, the ketothiolasephaA from R. eutropha combines two molecules of acetyl-CoA to formacetoacetyl-CoA (Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)).Similarly, a β-keto thiolase (bktB) has been reported to catalyze thecondensation of acetyl-CoA and propionyl-CoA to form β-ketovaleryl-CoA(Slater et al., J. Bacteriol. 180:1979-1987 (1998)) in R. eutropha. Theprotein sequences for the above-mentioned gene products are well knownin the art and can be accessed in the public databases such as GenBankusing the following accession numbers.

Gene name GenBank Accession # Organism paaJ NP_415915.1 Escherichia colipcaF AAL02407 Pseudomonas knackmussii (B13) phaD AAC24332.1 Pseudomonasputida paaE ABF82237.1 Pseudomonas fluorescens

These sequences can be used to identify homologue proteins in GenBank orother databases through sequence similarity searches, for example,BLASTp. The resulting homologue proteins and their corresponding genesequences provide additional exogenous DNA sequences for transformationinto E. coli or other microorganisms to generate production hosts.

For example, orthologs of paaJ from Escherichia coli K12 can be foundusing the following GenBank accession numbers:

YP_001335140.1 Klebsiella pneumoniae YP_001479310.1 Serratiaproteamaculans AAC24332.1 Pseudomonas putida

Example orthologs of pcaF from Pseudomonas knackmussii can be foundusing the following GenBank accession numbers:

AAD22035.1 Streptomyces sp. 2065 AAN67000.1 Pseudomonas putidaABJ15177.1 Pseudomonas aeruginosa

Additional native candidate genes for the ketothiolase step include atoBwhich can catalyze the reversible condensation of 2 acetyl-CoA molecules(Sato et al., J. Biosci. Bioengineer. 103:38-44 (2007)), and its homologyqeF. Non-native gene candidates include phaA (Sato et al., supra, 2007)and bktB (Slater et al., J. Bacteriol. 180:1979-1987 (1998)) from R.eutropha, and the two ketothiolases, thiA and thiB, from Clostridiumacetobutylicum (Winzer et al., J. Mol. Microbiol. Biotechnol. 2:531-541(2000)). The protein sequences for each of these exemplary gene productscan be found using the following GenBank accession numbers:

atoB NP_416728.1 Escherichia coli yqeF NP_417321.2 Escherichia coli phaAYP_725941 Ralstonia eutropha bktB AAC38322.1 Ralstonia eutropha thiANP_349476.1 Clostridium acetobutylicum thiB NP_149242.1 Clostridiumacetobutylicum

It is less desirable to use the thiolase-encoding genes fadA and fadB,genes in fatty acid degradation pathway in E. coli, in this exemplarypathway. These genes form a complex that encodes for multipleactivities, most of which are not desired in this pathway.

Referring to FIG. 3, step 2 involves 3-oxoadipyl-CoA transferase. Inthis step, 3-oxoadipate is formed by the transfer of the CoA group from3-oxoadipyl-CoA to succinate. This activity is reported in a two-unitenzyme encoded by pcaI and pcaJ in Pseudomonas (Kaschabek et al., J.Bacteriol. 184:207-215 (2002)). This enzyme catalyzes a reversibletransformation. The protein sequences of exemplary gene products forsubunit A of this complex can be found using the following GenBankaccession numbers:

pcaI AAN69545.1 Pseudomonas putida pcaI YP_046368.1 Acinetobacter sp.ADP1 pcaI NP_630776.1 Streptomyces coelicolor

The protein sequences of exemplary gene products for subunit B of thiscomplex can be found using the following GenBank accession numbers:

pcaJ NP_746082.1 Pseudomonas putida pcaJ NP_630775.1 Streptomycescoelicolor pcaJ AAC37147.1 Acinetobacter sp. ADP1

Referring to FIG. 3, step 3 involves 3-oxoadipate reductase. E. coli hasseveral candidate alcohol dehydrogenases; two that have analogousfunctions are malate dehydrogenase (mdh) and lactate dehydrogenase(ldhA). While it has not been shown that these two enzymes have broadsubstrate specificities in E. coli, lactate dehydrogenase from Ralstoniaeutropha has been shown to demonstrate high activities on substrates ofvarious chain lengths such as lactate, 2-oxobutyrate, 2-oxopentanoateand 2-oxoglutarate (Steinbuchel and Schlegel, Eur. J. Biochem.130:329-334 (1983)). An additional non-native enzyme candidate for thisstep is the mitochondrial 3-hydroxybutyrate dehydrogenase (bdh) from thehuman heart which has been cloned and characterized (Marks et al., J.Biol. Chem. 267:15459-15463 (1992)). This enzyme is particularlyinteresting in that it is a dehydrogenase that operates on a3-hydroxyacid. Given that dehydrogenases are typically reversible, it isexpected that this gene product, or a homlog thereof, will be capable ofreducing a 3-oxoacid, for example, 3-oxoadipate, to the corresponding3-hydroxyacid, for example, 3-hydroxyadipate. The protein sequences foreach of these exemplary gene products can be found using the followingGenBank accession numbers:

mdh AAC76268.1 Escherichia coli ldhA NP_415898.1 Escherichia coli ldhYP_725182.1 Ralstonia eutropha bdh AAA58352.1 Homo sapiens

Referring to FIG. 3, step 4 involves 3-hydroxyadipate dehydratase. Inthis reaction, 3-hydroxyadipate is dehydrated to hexa-2-enedioate.Although no direct evidence for this enzymatic transformation has beenidentified, most dehydratases catalyze the α,β-elimination of water.This involves activation of the α-hydrogen by an electron-withdrawingcarbonyl, carboxylate, or CoA-thiol ester group and removal of thehydroxyl group from the β-position (Martins et al., Proc. Natl. Acad.Sci. USA 101:15645-15649 (2004); Buckel and Golding, FEMS Microbiol.Rev. 22:523-541 (1998)). The protein sequences for exemplary geneproducts can be found using the following GenBank accession numbers:

acnA P25516.3 Escherichia coli fumB P14407.2 Escherichia coli ilvDAAA24013.1 Escherichia coli

Other good candidates for carrying out this function are the serinedehydratases. These enzymes catalyze a very similar transformation inthe removal of ammonia from serine as required in this dehydration step.The protein sequence for exemplary gene product can be found using thefollowing GenBank accession number:

dsdA P00926 Escherichia coli

Non-native gene candidates for this transformation have been identifiedas well. For example, the multi-subunit L-serine dehydratase fromPeptostreptococcus asaccharolyticus was shown to complement an E. colistrain deficient in L-serine dehydratase activity (Hofmeister et al., J.Bacteriol. 179:4937-4941 (1997)). Further, a putative2-(hydroxymethyl)glutarate dehydratase, encoded by the gene hmd inEubacterium barkeri shows similarity to both α- and β-subunits of[4Fe-4S]-containing bacterial serine dehydratases (Alhapel et al., Proc.Natl. Acad. Sci. USA 103:12341-12346 (2006)). The protein sequence forexemplary gene product can be found using the following GenBankaccession number:

hmd ABC88407.1 Eubacterium barkeri

Referring to FIG. 3, step 5 involves 2-enoate reductase. The final stepin the 3-oxoadipate pathway is reduction of the double bond inhexa-3-enedioate to form adipate. Biochemically, this transformation canbe catalyzed by 2-enoate reductase (EC 1.3.1.31) known to catalyze theNADH-dependent reduction of a wide variety of α,β-unsaturated carboxylicacids and aldehydes (Rohdich et al., J. Biol. Chem. 276:5779-5787(2001)). This enzyme is encoded by enr in several species of Clostridia(Giesel and Simon, Arch. Microbiol. 135:51-57 (1983)) including C.tyrobutyricum and C. thermoaceticum (now called Moorella thermoaceticum)(Rohdich, et al., J. Biol. Chem. 276:5779-5787 (2001)). In the recentlypublished genome sequence of C. kluyveri, 9 coding sequences for enoatereductases have been reported, out of which one has been characterized(Seedorf et al., Proc. Natl. Acad. Sci. USA 105:2128-2133 (2008)). Theenr genes from both C. tyrobutyricum and C. thermoaceticum have beencloned and sequenced and show 59% identity to each other. The formergene is also found to have approximately 75% similarity to thecharacterized gene in C. kluyveri (Giesel and Simon, Arch. Microbiol.135:51-57 (1983)). It has been reported based on these sequence resultsthat enr is very similar to the dienoyl CoA reductase in E. coli (fadH)(Rohdich et al., J. Biol. Chem. 276:5779-5787 (2001)). Several genecandidates thus exist for catalyzing this last step in the 3-oxoadipatepathway and have been listed below. The C. thermoaceticum enr gene hasalso been expressed in an enzymatically active form in E. coli (Rohdichet al., supra, 2001). The protein sequences for exemplary gene productscan be found using the following GenBank accession numbers:

fadH NP_417552.1 Escherichia coli enr ACA54153.1 Clostridium botulinumA3 str enr CAA71086.1 Clostridium tyrobutyricum enr CAA76083.1Clostridium kluyveri

The above description provides an exemplary adipate synthesis pathway byway of an 3-oxoadipate pathway.

Example IV Preparation of an Adipate Producing Microbial Organism Havinga 3-Oxoadipate Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using the 3-oxoadipate pathway.

Escherichia coli is used as a target organism to engineer the3-oxoadipate pathway as shown in FIG. 3. E. coli provides a good hostfor generating a non-naturally occurring microorganism capable ofproducing adipate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleicacids encoding the enzymes utilized in the 3-oxoadipate pathway areexpressed in E. coli using well known molecular biology techniques (see,for example, Sambrook, supra, 2001; Ausubel supra, 1999). In particular,the paaJ (NP_415915.1), pcaIJ (AAN69545.1 and NP_746082.1), and bdh(AAA58352.1) genes encoding the succinyl-CoA:acetyl-CoA acyltransferase, 3-oxoadipyl-CoA transferase, and 3-oxoadipate reductaseactivities, respectively, are cloned into the pZE13 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. In addition, the acnA(P25516.3) and enr (ACA54153.1) genes encoding 3-hydroxyadipatedehydratase and 2-enoate reductase activities, respectively, are clonedinto the pZA33 vector (Expressys, Ruelzheim, Germany) under the PA1/lacOpromoter. The two sets of plasmids are transformed into E. coli strainMG1655 to express the proteins and enzymes required for adipatesynthesis via the 3-oxoadipate pathway.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the3-oxoadipate pathway genes for adipate synthesis is corroborated usingmethods well known in the art for determining polypeptide expression orenzymatic activity, including for example, Northern blots, PCRamplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce adipate is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of adipate. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of adipate.Adaptive evolution also can be used to generate better producers of, forexample, the acetyl-CoA and succinyl-CoA intermediates or the adipateproduct. Adaptive evolution is performed to improve both growth andproduction characteristics (Fong and Palsson, Nat. Genet. 36:1056-1058(2004); Alper et al., Science 314:1565-1568 (2006)). Based on theresults, subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the adipate producer to further increaseproduction.

For large-scale production of adipate, the 3-oxoadipatepathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing the culture vessel, forexample, flasks can be sealed with a septum and crimp-cap. Microaerobicconditions also can be utilized by providing a small hole in the septumfor limited aeration. The pH of the medium is maintained at around a pHof 7 by addition of an acid, such as H₂SO₄. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm) and the glucose uptake rate by monitoring carbon source depletionover time. Byproducts such as undesirable alcohols, organic acids, andresidual glucose can be quantified by HPLC (Shimadzu), for example,using an Aminex® series of HPLC columns (for example, HPX-87 series)(BioRad), using a refractive index detector for glucose and alcohols,and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng.775-779 (2005)).

This example describes the preparation of an adipate-producing microbialorganism containing a 3-oxidoadipate pathway.

Example V Adipate Synthesis Via cis,cis-Muconic Acid

This example describes an adipate synthesis pathway previously described(see Niu et al., Biotechnol. Prog. 18(2): p. 201-11. 2002; Frost et al.,U.S. Pat. No. 5,487,987, issued Jan. 30, 1996).

Adipate synthesis via a combined biological and chemical conversionprocess has been previously described. (Niu et al., Biotechnol. Prog.18:201-211 (2002)) and is shown in FIG. 5. This method is furtherdescribed in U.S. Pat. No. 5,487,987. Adipate synthesis through thisroute entails introduction of three heterologous genes into E. coli thatcan convert dehydroshikimate into cis,cis-muconic acid (Niu et al.,supra, 2002). A final chemical hydrogenation step leads to the formationof adipic acid. In this step, the pretreated fermentation broth thatcontained 150 mM cis,cis-muconate was mixed with 10% platinum (Pt) onactivated carbon. The hydrogenation reaction was carried out at 3400 KPaof hydrogen pressure for two and a half hour at 250° C. with stirring.The calculated adipate yields are shown in Table 3 assuming either anenzymatic or chemical catalysis step is utilized to convertcis,cis-muconate into adipate. Under aerobic conditions, an 85% molaryield of adipate can be obtained if a chemical reaction is employed forhydrogenation and a 75% molar yield is obtained if an NADH-basedhydrogenase is used.

TABLE 3 The maximum theoretical yields of adipate per mole of glucoseusing the using the cis,cis-muconic acid pathway. Final step Final stepenzymatic chemical hydrogenation Aerobic Anaerobic Aerobic AnaerobicAdipate Yield 0.75 0.00 0.85 0.00

Although this is an exemplary method, there are disadvantages of thismethod compared to others, such as those described in Examples I-IV. Forexample, the first limitation of this method is the lower theoreticalyields compared to the reverse adipate degradation and 3-oxoadipatepathways. The second limitation is that the ATP yields of this pathwayare negligible. A third limitation of this pathway is that it involves adioxygenase, necessitating a supply of oxygen to the bioreactor andprecluding the option of anaerobic fermentation.

The above description provides an exemplary adipate synthesis pathway byway of a cis,cis-muconic acid pathway.

Example VI Adipate Synthesis Via Alpha-Ketoadipate

This example describes an exemplary adipate synthesis pathway via analpha-ketoadipate pathway.

Alpha-keto adipate is a known intermediate in lysine biosynthesis in S.cerevisiae, and this information was used to identify an additionalpathway for adipic acid biosynthesis (see FIG. 6). Conversion ofalpha-ketoglutarate to alpha-ketoadipate is catalyzed by homocitratesynthase, homoaconitase, and homoisocitrate dehydrogenase as indicatedby dashed arrows in FIG. 6. Conversion of alpha-ketoadipate intoalpha-hydroxyadipate can be catalyzed by 2-ketoadipate reductase, anenzyme reported to be found in rat and in human placenta (Suda et al.,Arch. Biochem. Biophys. 176:610-620 (1976); Suda et al., Biochem.Biophys. Res. Commun. 77:586-591 (1977). Subsequent steps involve adehydratase for the conversion of alpha-hydroxyadipate intohexa-2-enedioate followed by its reduction to adipic acid. This laststep can be catalyzed either by an enzyme or can take place through achemical reaction as described in Example II. Genes encoding the enzymesfor the alpha-ketoadipate pathway are identified as described inExamples I-IV.

The adipate yields associated with this pathway are shown in Table 4.Because of the loss of two CO₂ molecules during the conversion ofacetyl-CoA to adipate, only 67% of the glucose can be converted intoadipate. This is reflected in the molar yields for this pathway underaerobic conditions. The yields are further reduced in the absence ofoxygen uptake. Also since the maximum ATP yields under anaerobicconditions are negligible, the engineered organism will have to utilizeadditional substrate to form energy for cell growth and maintenanceunder such conditions.

TABLE 4 The maximum theoretical yields of adipate and the associated ATPyields per mole of glucose using the using the alpha-ketoadipatepathway. Final step Final step enzymatic chemical hydrogenation AerobicAnaerobic Aerobic Anaerobic Adipate Yield 0.67 0.45 0.67 0.40 Max ATPyield @ 6.17 0.00 7.50 0.00 max adipate yield

The above description provides an exemplary adipate synthesis pathway byway of an alpha-ketoadipate pathway.

Example VII Adipate Synthesis Via Lysine Degradation

This example describes an exemplary adipate synthesis pathway via alysine degradation pathway.

Two additional pathways for adipate synthesis rely on lysine degradationto form adipate. One pathway starts from alpha-ketoglutarate to formlysine (pathway non-native to E. coli and found in S. cerevisiae), andthe other uses aspartate as a starting point for lysine biosynthesis(pathway native to E. coli). FIG. 7 shows adipate formation from lysine.The maximum theoretical yields for adipate, both in the presence andabsence of oxygen, using the E. coli stoichiometric model are shown inTables 5 and 6, with alpha-ketoglutarate and aspartate as the respectivestarting points for lysine. The maximum ATP yields accompanying thesetheoretical yields were also calculated and are shown in the sametables. These yields are lower in comparison to the other pathwaysdescribed in Examples I-IV. Genes encoding the enzymes for thealpha-ketoadipate pathway are identified as described in Examples I-IV.

TABLE 5 The maximum theoretical yield of adipate and the accompanyingATP yield per mole of glucose assuming the lysine biosynthesis pathwaywith alpha-ketoglutarate as a starting point. Aerobic Anaerobic AdipateYield 0.40 0.20 Max ATP yield @ max adipate yield 5.60 0.00

TABLE 6 The maximum theoretical yield of adipate and the accompanyingATP yield per mole of glucose assuming the lysine biosynthesis pathwaywith aspartate as a starting point. Aerobic Anaerobic Adipate Yield 0.500.34 Max ATP yield @ max adipate yield 0.50 0.04

The above description provides an exemplary adipate synthesis pathway byway of a lysine degradation pathway.

Example VIII Production of Caprolactam and 6-Aminocaproic Acid ViaAdipyl-CoA

This example describes an exemplary caprolactam and/or 6-aminocaproicacid synthesis pathway via an adipyl-CoA pathway.

An exemplary pathway for forming caprolactam and/or 6-aminocaproic acidusing adipyl-CoA as the precursor is shown in FIG. 8. The pathwayinvolves a CoA-dependant aldehyde dehydrogenase that can reduceadipyl-CoA to adipate semialdehyde and a transaminase or 6-aminocaproatedehydrogenase that can transform this molecule into 6-aminocaproic acid.The terminal step that converts 6-aminocaproate into caprolactam can beaccomplished either via an amidohydrolase or via chemical conversion(Guit and Buij s, U.S. Pat. No. 6,353,100, issued Mar. 7, 2002; Wolterset al., U.S. Pat. No. 5,700,934, issued Dec. 23, 1997; Agterberg et al.,U.S. Pat. No. 6,660,857, issued Dec. 9, 2003). The maximum theoreticalyield of caprolactam was calculated to be 0.8 mole per mole glucoseconsumed (see Table 7) assuming that the reverse adipate degradationpathway was complemented with the reaction scheme shown in FIG. 8. Thepathway is favorable energetically as up to 0.78 moles of ATP are formedper mole of glucose consumed at the maximum theoretical yield ofcaprolactam. The ATP yield can be further improved to 1.63 moles of ATPproduced per mole of glucose if phosphoenolpyruvate carboxykinase (PPCK)is assumed to function in the ATP-generating direction towardsoxaloacetate formation.

The final amidohydrolase step is energetically and redox neutral, andthus the product and ATP molar yields associated with 6-aminocaproicacid production are equivalent to those associated with caprolactamproduction. Thus one can alternatively envision a microorganism andassociated fermentation process that forms 6-aminocaproic acid insteadof caprolactam followed by an additional unit operation todehydrate/cyclize 6-aminocaproic acid to caprolactam.

TABLE 7 The maximum theoretical yield of caprolactam and theaccompanying ATP yield per mole of glucose assuming that the reversefatty acid degradation pathway is complemented with the reaction schemefrom FIG. 8. Aerobic Anaerobic Caprolactam Yield 0.80 0.80 Max ATP yield@ max Caprolactam yield 0.78 0.78 Max ATP yield @ max Caprolactam yield1.63 1.63 PPCK assumed

Successfully engineering this pathway involves identifying anappropriate set of enzymes with sufficient activity and specificity.This entails identifying an appropriate set of enzymes, cloning theircorresponding genes into a production host, optimizing fermentationconditions, and assaying for product formation following fermentation.To engineer a production host for the production of 6-aminocaproic acidor caprolactam, one or more exogenous DNA sequence(s) can be expressedin a host microorganism. In addition, the microorganism can haveendogenous gene(s) functionally deleted. These modifications will allowthe production of 6-aminocaproate or caprolactam using renewablefeedstock.

Below is described a number of biochemically characterized candidategenes capable of encoding enzymes that catalyze each step of thecaprolactam formation pathway described in FIG. 8. Although describedfor E. coli, one skilled in the art can apply these teachings to anyother suitable host organism. Specifically, the genes listed are nativeto E. coli or are genes in other organisms that can be applied tocatalyze the appropriate transformations when properly cloned andexpressed.

Referring to FIG. 8, step 1 involves CoA-dependant aldehydedehydrogenase. Exemplary genes that encode enzymes for catalyzing thereduction of an acyl-coA to its corresponding aldehyde include theAcinetobacter calcoaceticus acr1 encoding a fatty acyl-CoA reductase(Reiser and Somerville, J. Bacteriol. 179:2969-2975 (1997)), theAcinetobacter sp. M-1 fatty acyl-CoA reductase (Ishige et al., Appl.Environ. Microbiol. 68:1192-1195 (2002)) and the sucD gene fromClostridium kluyveri (Sohling and Gottschalk, J. Bacteriol. 178:871-880(1996)), which can convert succinyl-CoA to succinate semialdehyde.

Gene name GenBank Accession # Organism acr1 YP_047869.1 Acinetobactercalcoaceticus BAB85476.1 Acinetobacter sp. Strain M-1 sucD P38947.1Clostridium kluyveri

Referring to FIG. 8, step 2 involves transaminase. The second step inthe pathway is conversion of the 6-aldehyde to an amine. Thistransformation can likely be accomplished by gamma-aminobutyratetransaminase (GABA transaminase), a native enzyme encoded by gabT thattransfers an amino group from glutamate to the terminal aldehyde ofsuccinyl semialdehyde (Bartsch et al., J. Bacteriol. 172:7035-7042(1990)). GABA transaminases in Mus musculus, Pseudomonas fluorescens,and Sus scrofa have been shown to react with 6-aminocaproic acid(Cooper, Methods Enzymol. 113:80-82 (1985); Scott and Jakoby, J. Biol.Chem. 234:932-936 (1959)). The protein sequences for exemplary geneproducts can be found using the following GenBank accession numbers:

gabT NP_417148.1 Escherichia coli abat NP_766549.2 Mus musculus gabTYP_257332.1 Pseudomonas fluorescens abat NP_999428.1 Sus scrofa

Referring to FIG. 8, step 2 can alternatively involve 6-aminocaproatedehydrogenase which comprises the reductive amination of adipatesemialdehyde to form 6-aminocaproate. This transformation can beaccomplished by lysine-6-dehydrogenase, which naturally convertsL-lysine to 2-aminoadipate-6-semialdehyde. Exemplary enzymes can befound in Geobacillus stearothermophilus (Heydari et al., Appl. Environ.Microbiol. 70(2):937-942 (2004)), Agrobacterium tumefaciens (Hashimotoet al., J. Biochem. (Tokyo), 106(1):76-80 (1989); Misono et al., J.Biochem. (Tokyo), 105(6):1002-1008 (1989)), and Achromobacterdenitrificans (Ruldeekulthamrong et al., BMB Reports 790-795 (2008)).

lysDH BAB39707 Geobacillus stearothermophilus lysDH NP_353966Agrobacterium tumefaciens lysDH AAZ94428 Achromobacter denitrificans

Referring to FIG. 8, step 3 involves amidohydrolase. The final step ofcaprolactam synthesis is cyclization of 6-aminocaproic acid. Thistransformation has not been characterized enzymatically but it is verysimilar to the cyclization of lysine by D-lysine lactamase (EC 3.5.2.11)from Cryptococcus laurentii (Fukumura et al., FEBS Lett. 89:298-300(1978)). However, the protein and nucleotide sequences of this enzymeare not currently known and, so far, lysine lactamase activity has notbeen demonstrated in other organisms.

Plasmids contained in several strains of Pseudomonas sp. isolated fromsoil have been shown to confer ability to grow on caprolactam as a solecarbon source (Boronin et al., FEMS Microbiol. Lett. 22:167-170 (1984));however, associated gene or protein sequences have not been associatedwith this function to date.

The most closely related candidate enzyme with available sequenceinformation is 6-aminohexanoate-cyclic dimer hydrolase, which has beencharacterized in Pseudomonas sp. and Flavobacterium sp. The nylB geneproduct from Pseudomonas sp NK87 was cloned and expressed in E. coli(Kanagawa et al., J. Gen. Microbiol. 139:787-795 (1993)). The substratespecificity of the enzyme was tested in Flavobacterium sp K172 and wasshown to react with higher-order oligomers of 6-aminohexanoate but notcaprolactam (Kinoshita et al., Eur. J. Biochem. 116:547-551 (1981)). Thereversibility and ability of 6-aminohexanoate dimer hydrolases in otherorganisms to react with the desired substrate in the direction ofinterest can be further tested. The protein sequences for exemplary geneproducts can be found using the following GenBank accession numbers:

nylB AAA24929.1 Pseudomonas sp NK87 nylB P13397 Flavobacterium sp K172nylB YP_949627.1 Arthrobacter aurescens TC1

The above description provides an exemplary pathway to producecaprolactam and/or 6-aminocaproic acid by way of an adipyl-CoA pathway.

Example IX Preparation of a 6-Aminocaproate or Caprolactam ProducingMicrobial Organism Having a 3-Oxoadipate Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using the reverse degradation pathway and convertingthe intracellular adipate to 6-aminocaproate and/or caprolactam.

Escherichia coli is used as a target organism to engineer the necessarygenes for adipate, 6-aminocaproate, and/or caprolactam synthesis (seeFIG. 2 and FIG. 8). E. coli provides a good host for generating anon-naturally occurring microorganism capable of producing adipate,6-aminocaproate, and/or caprolactam. E. coli is amenable to geneticmanipulation and is known to be capable of producing various products,like ethanol, acetic acid, formic acid, lactic acid, and succinic acid,effectively under anaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce 6-aminocaproateand/or caprolactam, nucleic acids encoding the enzymes utilized in thereverse adipate degradation pathway and 6-aminocaproate or caprolactamsynthesis pathways are expressed in E. coli using well known molecularbiology techniques (see, for example, Sambrook, supra, 2001; Ausubel,supra, 1999). In particular, the paaJ (NP_415915.1), paaH (NP_415913.1),and maoC (NP_415905.1) genes encoding the succinyl-CoA:acetyl-CoA acyltransferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyadipyl-CoAdehydratase activities, respectively, are cloned into the pZE13 vector(Expressys, Ruelzheim, Germany) under the PA1/lacO promoter. Inaddition, the bcd (NP_349317.1), etfAB (349315.1 and 349316.1), andsucCD (NP_415256.1 and AAC73823.1) genes encoding5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetaseactivities, respectively, are cloned into the pZA33 vector (Expressys,Ruelzheim, Germany) under the PA1/lacO promoter. Lastly, the acr1(YP_047869.1), gabT (NP_417148.1), and nylB (AAA24929.1) genes encodingCoA-dependent aldehyde dehydrogenase, transaminase, and amidohydrolaseactivities are cloned into a third compatible plasmid, pZS23, under thePA1/lacO promoter. pZS23 is obtained by replacing the ampicillinresistance module of the pZS13 vector (Expressys, Ruelzheim, Germany)with a kanamycin resistance module by well-known molecular biologytechniques. The three sets of plasmids are transformed into E. colistrain MG1655 to express the proteins and enzymes required for6-aminocaproate and/or caprolactam synthesis.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the6-aminocaproate and caprolactam synthesis genes is corroborated usingmethods well known in the art for determining polypeptide expression orenzymatic activity, including for example, Northern blots, PCRamplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce 6-aminocaproate and/or caprolactam is confirmed usingHPLC, gas chromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional pathway for thesynthesis of 6-aminocaproate and/or caprolactam are further augmented byoptimization for efficient utilization of the pathway. Briefly, theengineered strain is assessed to determine whether any of the exogenousgenes are expressed at a rate limiting level. Expression is increasedfor any enzymes expressed at low levels that can limit the flux throughthe pathway by, for example, introduction of additional gene copynumbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of 6-aminocaproate and/or caprolactam.One modeling method is the bilevel optimization approach, OptKnock(Burgard et al., Biotechnol. Bioengineer. 84:647-657 (2003)), which isapplied to select gene knockouts that collectively result in betterproduction of 6-aminocaproate and/or caprolactam. Adaptive evolutionalso can be used to generate better producers of, for example, theacetyl-CoA and succinyl-CoA intermediates of the products. Adaptiveevolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the 6-aminocaproate and/or caprolactamproducer to further increase production.

For large-scale production of 6-aminocaproate and/or caprolactam, theabove organism is cultured in a fermenter using a medium known in theart to support growth of the organism under anaerobic conditions.Fermentations are performed in either a batch, fed-batch or continuousmanner. Anaerobic conditions are maintained by first sparging the mediumwith nitrogen and then sealing the culture vessel, for example, flaskscan be sealed with a septum and crimp-cap. Microaerobic conditions alsocan be utilized by providing a small hole in the septum for limitedaeration. The pH of the medium is maintained at around a pH of 7 byaddition of an acid, such as H₂SO₄. The growth rate is determined bymeasuring optical density using a spectrophotometer (600 nm) and theglucose uptake rate by monitoring carbon source depletion over time.Byproducts such as undesirable alcohols, organic acids, and residualglucose can be quantified by HPLC (Shimadzu), for example, using anAminex® series of HPLC columns (for example, HPX-87 series) (BioRad),using a refractive index detector for glucose and alcohols, and a UVdetector for organic acids (Lin et al., Biotechnol. Bioeng. 775-779(2005)).

Example X Adipate Synthesis Via 2-Hydroxyadipyl-CoA

This example describes two exemplary adipate synthesis pathwaysproceeding from alpha-ketoadipate and passing through a2-hydroxyadipyl-CoA intermediate.

As described in example VI, alpha-ketoadipate is a known intermediate inlysine biosynthesis that can be formed from alpha-ketoglutarate viahomocitrate synthase, homoaconitase, and homoisocitrate dehydrogenase.Alpha-ketoadipate can be converted to 2-hydroxyadipyl-CoA by the tworoutes depicted in FIG. 9. 2-hydroxyadipyl-CoA can be subsequentlydehydrated and reduced to adipyl-CoA which can then be converted toadipate as shown in FIG. 9. The maximum yield of adipate from glucosevia these pathways is 0.67 mol/mol.

Conversion of alpha-ketoadipate into 2-hydroxyadipate can be catalyzedby 2-ketoadipate reductase, an enzyme reported to be found in rat and inhuman placenta (Suda et al., Arch. Biochem. Biophys. 176:610-620 (1976);Suda et al., Biochem. Biophys. Res. Commun. 77:586-591 (1977).Alternatively, enzymes capable of reducing alpha-ketoglutarate to2-hydroxyglutarate may also show activity on alpha-ketoadipate, which isonly one carbon atom longer. One such enzyme possessingalpha-ketoglutarate reductase activity is serA of Escherichia coli (Zhaoand Winkler, J. Bacteriol. 178(1):232-9 (1996)). Additional exemplaryenzymes can be found in Arabidopsis thaliana (Ho, et al., J. Biol. Chem.274(1):397-402 (1999)) and Haemophilus influenzae.

serA NP_417388.1 Escherichia coli PGDH NP_564034 Arabidopsis thalianaserA P43885 Haemophilus influenzae

Referring to FIG. 9, 2-hydroxyadipate can likely be converted to2-hydroxyadipyl-CoA by the synthetases, transferases,phosphotransadipylases and kinases described in example I.Alternatively, enzymes with 2-hydroxyglutarate CoA-transferase orglutaconate CoA-transferase activity are likely suitable to transfer aCoA moiety to 2-hydroxyadipate. One example of such an enzyme is encodedby the gctA and gctB genes of Acidaminococcus fermentans (Buckel, etal., Eur. J. Biochem. 118(2):315-321 (1981); Mack, et al., Eur. J.Biochem. 226(1):41-51 (1994)). Similarly, synthetase, transferase, orphosphotransadipylase and kinase activities would be required to convertalpha-ketoadipate into alpha-ketoadipyl-CoA, as depicted in FIG. 9.Conversion of alpha-ketoadipyl-CoA to 2-hydroxyadipyl-CoA can be carriedout by an alpha-hydroxyacyl-CoA dehydrogenase enzyme. A similar activitywas reported in propionate-adapted E. coli cells whose extractscatalyzed the oxidation of lactyl-CoA to form pyruvyl-CoA (Megraw etal., J. Bacteriol. 90(4): 984-988 (1965)). Additional hydroxyacyl-CoAdehydrogenases were described in example I.

gctA Q59111 Acidaminococcus fermentans gctB Q59112 Acidaminococcusfermentans

The dehydration of 2-hydroxyadipyl-CoA to form 5-carboxy-2-pentenoyl-CoAcan be carried out by a 2-hydroxyacyl-CoA dehydratase. A2-hydroxyglutaryl-CoA dehydratase system has been characterized inAcidaminococcus fermentans and requires both the hgdA and hgdB subunitsand the activator protein, hgdC, for optimal activity (Dutscho et al.,Eur. J. Biochem. 181(3):741-746 (1989); Locher et al. J. Mol. Biol.307(1):297-308; Muller and Buckel, Eur. J. Biochem. 230(2):698-704(2001); Schweiger et al. Eur. J. Biochem. 169(2):441-448 (1987)). Thisenzyme system is similar in mechanism to the lactoyl-CoA dehydratasefrom Clostridium propionicum (Hofmeister and Buckel, Eur. J. Biochem.206(2):547-552 (1992); Kuchta and Abeles, J. Biol. Chem.260(24):13181-13189 (1985)). Homologs to hgdA, hgdB, and hgdC exist inseveral organisms.

hgdA P11569 Acidaminococcus fermentans hgdB P11570 Acidaminococcusfermentans hgdC P11568 Acidaminococcus fermentans hgdA ZP_03731126.1Clostridium sp. M62/1 hgdB ZP_03731125.1 Clostridium sp. M62/1 hgdCZP_03731127.1 Clostridium sp. M62/1 hgdA NP_603114.1 Fusobacteriumnucleatum ATCC 25586 hgdB NP_603115.1 Fusobacterium nucleatum ATCC 25586hgdC NP_603113.1 Fusobacterium nucleatum ATCC 25586Conversion of 5-carboxy-2-pentenoyl-CoA to adipate is carried out by theenzymes described in Example I.

The above description provides an exemplary adipate synthesis pathway byway of a 2-hydroxyadipyl-CoA pathway.

Example XI Preparation of an Adipate Producing Microbial Organism Havinga 2-Hydroxyadipyl-CoA Pathway

This example describes the generation of a microbial organism capable ofproducing adipate using a 2-hydroxyadipyl-CoA pathway.

Escherichia coli is used as a target organism to engineer the necessarygenes for adipate synthesis (see FIG. 9). E. coli provides a good hostfor generating a non-naturally occurring microorganism capable ofproducing adipate. E. coli is amenable to genetic manipulation and isknown to be capable of producing various products, like ethanol, aceticacid, formic acid, lactic acid, and succinic acid, effectively underanaerobic or microaerobic conditions.

To generate an E. coli strain engineered to produce adipate, nucleicacids encoding the enzymes utilized in a 2-hydroxyadipyl-CoA to adipatepathway are expressed in E. coli using well known molecular biologytechniques (see, for example, Sambrook, supra, 2001; Ausubel, supra,1999). In particular, the serA (NP_417388.1), gctA (Q59111), and gctB(Q59112) genes encoding the 2-hydroxyadipate dehydrogenase and2-hydroxyadipyl-CoA:acetyl-CoA transferase activities, respectively, arecloned into the pZE13 vector (Expressys, Ruelzheim, Germany) under thePA1/lacO promoter. In addition, the hgdA (P11569), hgdB (P11570), andhgdC (P11568) genes encoding 2-hydroxyadipyl-CoA dehydratase activity,respectively, are cloned into the pZA33 vector (Expressys, Ruelzheim,Germany) under the PA1/lacO promoter. Further, the bcd (NP_349317.1),etfAB (349315.1 and 349316.1), and sucCD (NP_415256.1 and AAC73823.1)genes encoding 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoAsynthetase activities are cloned into a third compatible plasmid, pZS23,under the PA1/lacO promoter. pZS23 is obtained by replacing theampicillin resistance module of the pZS13 vector (Expressys, Ruelzheim,Germany) with a kanamycin resistance module by well-known molecularbiology techniques. The three sets of plasmids are transformed into E.coli strain MG1655 to express the proteins and enzymes required foradipate synthesis.

The resulting genetically engineered organism is cultured in glucosecontaining medium following procedures well known in the art (see, forexample, Sambrook et al., supra, 2001). The expression of the2-hydroxyadipyl-CoA pathway genes for adipate synthesis is corroboratedusing methods well known in the art for determining polypeptideexpression or enzymatic activity, including for example, Northern blots,PCR amplification of mRNA, immunoblotting, and the like. Enzymaticactivities of the expressed enzymes are confirmed using assays specificfor the individual activities. The ability of the engineered E. colistrain to produce adipate is confirmed using HPLC, gaschromatography-mass spectrometry (GCMS) and/or liquidchromatography-mass spectrometry (LCMS).

Microbial strains engineered to have a functional adipate synthesispathway are further augmented by optimization for efficient utilizationof the pathway. Briefly, the engineered strain is assessed to determinewhether any of the exogenous genes are expressed at a rate limitinglevel. Expression is increased for any enzymes expressed at low levelsthat can limit the flux through the pathway by, for example,introduction of additional gene copy numbers.

To generate better producers, metabolic modeling is utilized to optimizegrowth conditions. Modeling is also used to design gene knockouts thatadditionally optimize utilization of the pathway (see, for example, U.S.patent publications US 2002/0012939, US 2003/0224363, US 2004/0029149,US 2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,and U.S. Pat. No. 7,127,379). Modeling analysis allows reliablepredictions of the effects on cell growth of shifting the metabolismtowards more efficient production of adipate. One modeling method is thebilevel optimization approach, OptKnock (Burgard et al., Biotechnol.Bioengineer. 84:647-657 (2003)), which is applied to select geneknockouts that collectively result in better production of adipate.Adaptive evolution also can be used to generate better producers of, forexample, the alpha-ketoadipate intermediate or the adipate product.Adaptive evolution is performed to improve both growth and productioncharacteristics (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004);Alper et al., Science 314:1565-1568 (2006)). Based on the results,subsequent rounds of modeling, genetic engineering and adaptiveevolution can be applied to the adipate producer to further increaseproduction.

For large-scale production of adipate, the 2-hydroxyadipyl-CoApathway-containing organism is cultured in a fermenter using a mediumknown in the art to support growth of the organism under anaerobicconditions. Fermentations are performed in either a batch, fed-batch orcontinuous manner. Anaerobic conditions are maintained by first spargingthe medium with nitrogen and then sealing the culture vessel, forexample, flasks can be sealed with a septum and crimp-cap. Microaerobicconditions also can be utilized by providing a small hole in the septumfor limited aeration. The pH of the medium is maintained at around a pHof 7 by addition of an acid, such as H₂SO₄. The growth rate isdetermined by measuring optical density using a spectrophotometer (600nm) and the glucose uptake rate by monitoring carbon source depletionover time. Byproducts such as undesirable alcohols, organic acids, andresidual glucose can be quantified by HPLC (Shimadzu), for example,using an Aminex® series of HPLC columns (for example, HPX-87 series)(BioRad), using a refractive index detector for glucose and alcohols,and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng.775-779 (2005)).

This example describes the preparation of an adipate-producing microbialorganism containing a 2-hydroxyadipyl-CoA pathway.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains. Althoughthe invention has been described with reference to the examples providedabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention.

What is claimed is:
 1. A non-naturally occurring microbial organism,comprising a microbial organism having an adipate pathway comprising atleast one exogenous nucleic acid encoding an adipate pathway enzymeexpressed in a sufficient amount to produce adipate, said adipatepathway comprising alpha-ketoadipyl-CoA synthetase,phosphotransketoadipylase/alpha-ketoadipate kinase oralpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydrogenase; 2-hydroxyadipyl-CoA dehydratase;5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferaseor adipyl-CoA hydrolase.
 2. The non-naturally occurring microbialorganism of claim 1, wherein said microbial organism comprises twoexogenous nucleic acids each encoding an adipate pathway enzyme.
 3. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism comprises three exogenous nucleic acids each encodingan adipate pathway enzyme.
 4. The non-naturally occurring microbialorganism of claim 1, wherein said microbial organism comprises fourexogenous nucleic acids each encoding an adipate pathway enzyme.
 5. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism comprises five exogenous nucleic acids each encodingan adipate pathway enzyme.
 6. The non-naturally occurring microbialorganism of claim 5, wherein said five exogenous nucleic acids encodealpha-ketoadipyl-CoA synthetase,phosphotransketoadipylase/alpha-ketoadipate kinase oralpha-ketoadipyl-CoA:acetyl-CoA transferase; 2-hydroxyadipyl-CoAdehydrogenase; 2-hydroxyadipyl-CoA dehydratase;5-carboxy-2-pentenoyl-CoA reductase; and adipyl-CoA synthetase,phosphotransadipylase/adipate kinase, adipyl-CoA:acetyl-CoA transferaseor adipyl-CoA hydrolase.
 7. The non-naturally occurring microbialorganism of claim 1, wherein said at least one exogenous nucleic acid isa heterologous nucleic acid.
 8. The non-naturally occurring microbialorganism of claim 1, wherein said non-naturally occurring microbialorganism is in a substantially anaerobic culture medium.
 9. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism is a species of bacteria, yeast or fungus.
 10. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism is a species of bacteria selected from the groupconsisting of Escherichia coli, Klebsiella oxytoca, Anaerobiospirillumsuccinicproducens, Actinobacillus succinogenes, Mannheimiasucciniproducens, Rhizobium etli, Bacillus subtilis, Corynebacteriumglutamicum, Gluconobacter oxydans, Zymomonas mobilis, Lactococcuslactis, Lactobacillus plantarum, Streptomyces coelicolor, Clostridiumacetobutylicum, Pseudomonas fluorescens and Pseudomonas putida.
 11. Thenon-naturally occurring microbial organism of claim 1, wherein saidmicrobial organism is a species of yeast or fungus selected from thegroup consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger and Pichia pastoris.
 12. The non-naturally occurringmicrobial organism of claim 11, wherein said microbial organism is aspecies of Saccharomyces cerevisiae.
 13. A method for producing adipate,comprising culturing the non-naturally occurring microbial organism ofclaim 1 under conditions and for a sufficient period of time to produceadipate.
 14. The method of claim 13, wherein said non-naturallyoccurring microbial organism is in a substantially anaerobic culturemedium.
 15. The method of claim 13, wherein said method furthercomprises separating the adipate from other components in the culture.16. The method of claim 15, wherein said separating comprisesextraction, continuous liquid-liquid extraction, pervaporation, membranefiltration, membrane separation, reverse osmosis, electrodialysis,distillation, crystallization, centrifugation, extractive filtration,ion exchange chromatography, size exclusion chromatography, absorptionchromatography or ultrafiltration.